Repair and regeneration of ocular tissue using postpartum-derived cells

ABSTRACT

Cells derived from postpartum umbilicus and placenta are disclosed. Pharmaceutical compositions, devices and methods for the regeneration or repair of ocular tissue using the postpartum-derived cells are also disclosed.

This a continuation in part of U.S. application Ser. No. 10/877,541,filed Jun. 25, 2004, now U.S. Pat. No. 7,413,734, issued Aug. 19, 2008,the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of cell-based or regenerativetherapy for ophthalmic diseases and disorders. In particular, theinvention provides pharmaceutical compositions, devices and methods forthe regeneration or repair of cells and tissues of the eye, usingpostpartum derived cells.

BACKGROUND

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

As a complex and sensitive organ of the body, the eye can experiencenumerous diseases and other deleterious conditions that affect itsability to function normally. Many of these conditions are associatedwith damage or degeneration of specific ocular cells, and tissues madeup of those cells. As one example, diseases and degenerative conditionsof the optic nerve and retina are the leading causes of blindnessthroughout the world. Damage or degeneration of the cornea, lens andassociated ocular tissues represent another significant cause of visionloss worldwide.

The retina contains seven layers of alternating cells and processes thatconvert a light signal into a neural signal. The retinal photoreceptorsand adjacent retinal pigment epithelium (RPE) form a functional unitthat, in many disorders, becomes unbalanced due to genetic mutations orenvironmental conditions (including age). This results in loss ofphotoreceptors through apoptosis or secondary degeneration, which leadsto progressive deterioration of vision and, in some instances, toblindness (for a review, see, e.g., Lund, R. D. et al. 2001, Progress inRetinal and Eye Research 20: 415-449). Two classes of ocular disordersthat fall into this pattern are age-related macular degeneration (AMD)and retinitis pigmentosa (RP).

AMD is the most common cause of vision loss in the United States inthose people whose ages are 50 or older, and its prevalence increaseswith age. The primary disorder in AMD appears to be due to RPEdysfunction and changes in Bruch's membranes, characterized by, amongother things, lipid deposition, protein cross-linking and decreasedpermeability to nutrients (see Lund et al., 2001 supra). A variety ofelements may contribute to macular degeneration, including geneticmakeup, age, nutrition, smoking and exposure to sunlight.

AMD is broadly divided into two types. In the exudative-neovascularform, or “wet” AMD, which accounts for 10% of all cases, abnormal bloodvessel growth occurs under the macula. These blood vessels leak fluidand blood into the retina and thus cause damage to the photoreceptors.The remaining 90% of AMD cases are the nonexudative, or “dry” form. Inthese patients there is a gradual disappearance of the retinal pigmentepithelium (RPE), resulting in circumscribed areas of atrophy. Sincephotoreceptor loss follows the disappearance of RPE, the affectedretinal areas have little or no visual function.

For example, Radeke, M. et al state “Macular degeneration is a blindingdisease caused by the death of the photoreceptor cells in that part ofthe retina known as the macula (Radeke M et al, 2007). Radeke, M. et alfurther state, “Photoreceptors are critically dependent upon the RPEcells for their own survival” (Radeke M et al, 2007).

Current therapies for AMD involve procedures, such as, for example,laser therapy and pharmacological intervention. By transferring thermalenergy, the laser beam destroys the leaky blood vessels under themacula, which slows the rate of vision loss. A disadvantage of thisapproach, however, is that the high thermal energy delivered by the beamalso destroys healthy tissue nearby. Neuroscience 4^(th) edition,(Purves, D, et al 2008) states “Currently there is no treatment for dryAMD.”

RPE transplantation, following excision of choroid neovascularization isalso one potential cell-based therapy for AMD. However, this has beenunsuccessful in humans. For example, Zarbin, M, 2003 states, “Withnormal aging, human Bruch's membrane, especially in the submacularregion, undergoes numerous changes (e.g., increased thickness,deposition of ECM and lipids, cross-linking of protein, non-enzymaticformation of advanced glycation end products). These changes andadditional changes due to AMD could decrease the bioavailability of ECMligands (e.g., laminin, fibronectin, and collagen IV) and cause theextremely poor survival of RPE cells in eyes with AMD. Thus, althoughhuman RPE cells express the integrins needed to attach to these ECMmolecules, RPE cell survival on aged submacular human Bruch's membraneis impaired”.

RP is mainly considered an inherited disease, with over 100 mutationsbeing associated with photoreceptor loss (see Lund et al., 2001, supra).Though the majority of mutations target photoreceptors, some affect RPEcells directly. Together, these mutations affect such processes asmolecular trafficking between photoreceptors and RPE cells andphototransduction, for example.

Other less common, but nonetheless debilitating retinopathies can alsoinvolve progressive cellular degeneration leading to vision loss andblindness. These include, for example, diabetic retinopathy andchoroidal neovascular membrane (CNVM).

Diabetic retinopathy may be classified as (1) non-proliferative orbackground retinopathy, characterized by increased capillarypermeability, edema, hemorrhage, microaneurysms, and exudates, or 2)proliferative retinopathy, characterized by neovascularization extendingfrom the retina to the vitreous, scarring, fibrous tissue formation, andpotential for retinal detachment.

In CNVM, abnormal blood vessels stemming from the choroid grow upthrough the retinal layers. The fragile new vessels break easily,causing blood and fluid to pool within the layers of the retina.

Damage or progressive degeneration of the optic nerve and related nervesof the eye constitutes another leading cause of vision loss andblindness. A prime example is glaucoma, a condition of the eye that ismade up of a collection of eye diseases that cause vision loss by damageto the optic nerve. Elevated intraocular pressure (IOP) due toinadequate ocular drainage is a primary cause of glaucoma, but it canalso develop in the absence of elevated IOP. Glaucoma can develop as theeye ages. It can also occur as the result of an eye injury,inflammation, tumor, or in advanced cases of cataract or diabetes, or itcan be caused by certain drugs, such as, for example, steroids. Theprimary features of the optic neuropathy in glaucoma includecharacteristic changes in the optic nerve head, a decrease in number ofsurviving retinal ganglion cells, and loss of vision. It has beenproposed that a cascade of events links degeneration of the optic nervehead with the slow death of retinal ganglion cells observed in thedisease, and that this cascade of events can be slowed or preventedthrough the use of neuroprotective agents (Osborne et al., 2003, Eur. J.Opthalmol. 13 (Supp 3): S19-S26).

Cellular damage and degenerative conditions also affect other parts ofthe eye. For example, cataracts result from gradual opacification of thecrystalline lens of the eye. It is believed that once begun, cataractdevelopment proceeds along one or more common pathways that culminate indamage to lens fibers. This condition is presently treated by surgicalremoval and replacement of the affected lens. Another example concernsthe cornea and surrounding conjuctiva that make up the ocular surface.The limbal epithelium, located between the cornea and the bulbarconjuctiva, contains corneal epithelial stem cells. Limbal epithelialcell deficiency (LECD) is a condition that occurs, for example, inStevens-Johnson syndrome and thermal or chemical burns. LECD often leadsto an imbalance between the corneal epithelium and the conjunctivalepithelium in which the cornea is covered by invading conjunctivalepithelial cells, which severely compromises the corneal surface andaffects visual acuity (Nakamura, T. & Kinoshita, S., 2003. Cornea 22(Supp. 1): S75-S80).

The recent advent of stem cell-based therapy for tissue repair andregeneration provides promising treatments for a number ofaforementioned cell-degenerative pathologies and other ocular disorders.Stem cells are capable of self-renewal and differentiation to generate avariety of mature cell lineages. Transplantation of such cells can beutilized as a clinical tool for reconstituting a target tissue, therebyrestoring physiologic and anatomic functionality. The application ofstem cell technology is wide-ranging, including tissue engineering, genetherapy delivery, and cell therapeutics, i.e., delivery ofbiotherapeutic agents to a target location via exogenously suppliedliving cells or cellular components that produce or contain those agents(For a review, see, for example, Tresco, P. A. et al., 2000, AdvancedDrug Delivery Reviews 42: 2-37).

An obstacle to realization of the therapeutic potential of stem celltechnology has been the difficulty in obtaining sufficient numbers ofstem cells. One source of stem cells is embryonic or fetal tissue.Embryonic stem and progenitor cells have been isolated from a number ofmammalian species, including humans, and several such cell types havebeen shown capable of self-renewal and expansion, as welldifferentiation into a variety of cell lineages. In animal modelsystems, embryonic stem cells have been reported to differentiate into aRPE cell phenotype, as well as to enhance the survival of hostphotoreceptors following transplantation (Haruta, M. et al., 2004,Investig. Opthalmol. Visual Sci. 45: 1020-1025; Schraermeyer, U. et al.,2001, Cell Transplantation 10: 673-680). But the derivation of stemcells from embryonic or fetal sources has raised many ethical issuesthat are desirable to avoid by identifying other sources of multipotentor pluripotent cells.

Adult tissue also can yield stem cells useful for cell-based oculartherapy. For instance, retinal and corneal stem cells themselves may beutilized for cell replacement therapy in the eye. In addition, neuralstem cells from the hippocampus have been reported to integrate with thehost retina, adopting certain neural and glial characteristics (seereview of Lund, R. L. et al., 2003, J. Leukocyte Biol. 74: 151-160).Neural stem cells prepared from fetal rat cortex were shown todifferentiate along an RPE cell pathway following transplantation intothe adult rat subretinal space (Enzmann, V. et al., 2003, Investig.Opthalmol. Visual Sci. 44: 5417-5422). Bone marrow stem cells have beenreported to differentiate into retinal neural cells and photoreceptorsfollowing transplantation into host retinas (Tomita, M. et al., 2002,Stem Cells 20: 279-283; Kicic, A. et al., 2003, J. Neurosci. 23:7742-7749). An ocular surface reconstruction in a rabbit model system,utilizing cultured mucosal epithelial stem cells, has also been reported(Nakamura, T. & Kinoshita, S., 2003, supra). While these reports showpromise for the use of adult progenitor and stem cells in cell-basedtherapy for the eye, it must be noted that adult stem cell populationsare comparatively rare and are often obtainable only by invasiveprocedures. Further, adult stem cells may have a more limited ability toexpand in culture than do embryonic stem cells.

Thus, a need exists for alternative sources of adequate supplies ofcells having the ability to support, augment or replace lost cellularfunction in the eye. A reliable, well-characterized and plentiful supplyof substantially homogeneous populations of such cells would be anadvantage in a variety of diagnostic and therapeutic applications inocular repair and regeneration, including drug screening assays, ex vivoor in vitro trophic support of ocular and other useful cell types, andin vivo cell-based therapy.

SUMMARY

This invention provides compositions and methods applicable tocell-based or regenerative therapy for ophthalmic diseases anddisorders. In particular, the invention features pharmaceuticalcompositions, devices and methods for the regeneration or repair ofocular tissue using postpartum-derived cells.

One aspect of the invention features an isolated postpartum-derivedcell, derived from human placental or umbilical cord tissuesubstantially free of blood, wherein the cell is capable of self-renewaland expansion in culture and has the potential to differentiate into acell of a neural phenotype; wherein the cell requires L-valine forgrowth and is capable of growth in at least about 5% oxygen. This cellfurther comprises one or more of the following characteristics: (a)potential for at least about 40 doublings in culture; (b) attachment andexpansion on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyomithine, vitronectin, or fibronectin; (c) production ofat least one of tissue factor, vimentin, and alpha-smooth muscle actin;(d) production of at least one of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, PD-L2and HLA-A,B,C; (e) lack of production of at least oneof CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, andHLA-DR,DP,DQ, as detected by flow cytometry; (f) expression of a gene,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an iliac crest bone marrow cell, is increased for at least oneof a gene encoding: interleukin 8; reticulon 1; chemokine (C—X—C motif)ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C—X—Cmotif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C—X—Cmotif) ligand 3; tumor necrosis factor, alpha-induced protein 3; C-typelectin superfamily member 2; Wilms tumor 1; aldehyde dehydrogenase 1family member A2; renin; oxidized low density lipoprotein receptor 1;Homo sapiens clone IMAGE:4179671; protein kinase C zeta; hypotheticalprotein DKFZp564F013; downregulated in ovarian cancer 1; and Homosapiens gene from clone DKFZp547k1113; (g) expression of a gene, whichrelative to a human cell that is a fibroblast, a mesenchymal stem cell,or an iliac crest bone marrow cell, is reduced for at least one of agene encoding: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C—X—C motif) ligand 12(stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeo box 2 (growth arrest-specific homeo box); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; disheveled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1;insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, beta 7; transcriptional co-activatorwith PDZ-binding motif (T AZ); sine oculis homeobox homolog 2(Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;early growth response 3; distal-less homeo box 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; transcriptionalco-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin;integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNAfull length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367protein; natriuretic peptide receptor C/guanylate cyclase C(atrionatriuretic peptide receptor C); hypothetical protein FLJ14054;Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222);BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);similar to neuralin 1; B cell translocation gene 1; hypothetical proteinFLJ23191; and DKFZp586L151; (h) secretion of at least one of MCP-1,IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, andTIMP1; and (i) lack of secretion of at least one of TGF-beta2, ANG2,PDGFbb, MIP1b, I309, MDC, and VEGF, as detected by ELISA.

In certain embodiments, the postpartum-derived cell is anumbilicus-derived cell. In other embodiments it is a placenta-derivedcell. In specific embodiments, the cell has all identifying features ofany one of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074);cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); cell type PLA071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7)(ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCCAccession No. PTA-6068).

In certain embodiments, postpartum-derived cells are isolated in thepresence of one or more enzyme activities comprising metalloproteaseactivity, mucolytic activity and neutral protease activity. Preferably,the cells have a normal karyotype, which is maintained as the cells arepassaged in culture. In preferred embodiments, the postpartum-derivedcells comprise each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A, B, C and does not comprise any of CD31, CD34, CD45, CD117, CD141,or HLA-DR, DP, DQ, as detected by flow cytometry.

Another aspect of the invention features a cell population comprisingthe postpartum-derived cells as described above. In one embodiment, thepopulation is a substantially homogeneous population of thepostpartum-derived cells. In a specific embodiment, the populationcomprises a clonal cell line of the postpartum-derived cells. In anotherembodiment, the population is a heterogeneous population comprising thepostpartum-derived cells and at least one other cell type. In certainembodiments, the other cell type is an astrocyte, oligodendrocyte,neuron, neural progenitor, neural stem cell, retinal epithelial stemcell, corneal epithelial stem cell or other multipotent or pluripotentstem cell. In other embodiments, the cell population is cultured incontact with one or more factors that stimulate stem celldifferentiation toward a neural or epithelial lineage.

Also featured in accordance with the present invention is a cell lysateprepared from postpartum-derived cells. The cell lysate may be separatedinto a membrane enriched fraction and a soluble cell fraction. Theinvention also features an extracellular matrix produced by thepostpartum-derived cells, as well as a conditioned medium in which thecells have been grown.

Another aspect of the invention features a method of treating a patienthaving an ocular degenerative condition, which comprises administeringto the patient multipotent or pluripotent cells isolated from apostpartum placenta or umbilical cord, in an amount effective to treatthe ocular degenerative condition. Preferably the cells arepostpartum-derived cells, as described above. In certain embodiments,the ocular degenerative condition is an acute ocular degenerativecondition such as brain trauma, optic nerve trauma or ocular lesion. Inother embodiments, it is a chronic or progressive degenerativecondition, such as macular degeneration, retinitis pigmentosa, diabeticretinopathy, glaucoma or limbal epithelial cell deficiency. In certainembodiments, the cells are induced in vitro to differentiate into aneural or epithelial lineage cells prior to administration.

In certain embodiments, the cells are administered with at least oneother cell type, such as an astrocyte, oligodendrocyte, neuron, neuralprogenitor, neural stem cell, retinal epithelial stem cell, cornealepithelial stem cell, or other multipotent or pluripotent stem cell. Inthese embodiments, the other cell type can be administeredsimultaneously with, or before, or after, the postpartum-derived cells.Likewise, in these and other embodiments, the cells are administeredwith at least one other agent, such as a drug for ocular therapy, oranother beneficial adjunctive agent such as an anti-inflammatory agent,anti-apoptotic agents, antioxidants or growth factors. In theseembodiments, the other agent can be administered simultaneously with, orbefore, or after, the postpartum cells.

In various embodiments, the cells are administered to the surface of aneye, or they are administered to the interior of an eye or to a locationin proximity to the eye, e.g., behind the eye. The cells can beadministered through a cannula or from a device implanted in thepatient's body within or in proximity to the eye, or they may beadministered by implantation of a matrix or scaffold containing thecells.

Another aspect of the invention features a pharmaceutical compositionfor treating a patient having an ocular degenerative condition,comprising a pharmaceutically acceptable carrier and multipotent orpluripotent cells isolated from a postpartum placenta or umbilical cordin an amount effective to treat the ocular degenerative condition.Preferably the postpartum cells are postpartum-derived cells asdescribed above. The ocular degenerative condition may be an acute,chronic or progressive condition. In certain embodiments, thecomposition comprises cells that have been induced in vitro todifferentiate into a neural or epithelial lineage cells prior toformulation of the composition.

In certain embodiments, the pharmaceutical composition comprises atleast one other cell type, such as an astrocyte, oligodendrocyte,neuron, neural progenitor, neural stem cell, retinal epithelial stemcell, corneal epithelial stem cell, or other multipotent or pluripotentstem cell. In these or other embodiments, the pharmaceutical compositioncomprises at least one other agent, such as a drug for treating theocular degenerative disorder or other beneficial adjunctive agents,e.g., anti-inflammatory agents, anti-apoptotic agents, antioxidants orgrowth factors.

In certain embodiments, the pharmaceutical compositions are formulatedfor administration to the surface of an eye. Alternatively, they can beformulated for administration to the interior of an eye or in proximityto the eye (e.g., behind the eye). The compositions also can beformulated as a matrix or scaffold containing the cells.

According to yet another aspect of the invention, a kit is provided fortreating a patient having an ocular degenerative condition. The kitcomprises a pharmaceutically acceptable carrier, a population ofmultipotent or pluripotent cells isolated from postpartum placenta orumbilicus, preferably the postpartum-derived cells described above, andinstructions for using the kit in a method of treating the patient. Thekit may also contain one or more additional components, such as reagentsand instructions for culturing the cells, or a population of at leastone other cell type, or one or more agents useful in the treatment of anocular degenerative condition.

According to another aspect of the invention, a method is provided fortreating a patient having an ocular degenerative condition, whichcomprises administering to the patient a preparation made frommultipotent or pluripotent cells isolated from a postpartum placenta orumbilical cord, in an amount effective to treat the ocular degenerativecondition, wherein the preparation comprises a cell lysate of the cells(or fraction thereof), or a conditioned medium in which the cells weregrown, or an extracellular matrix of the cells. Preferably, the cellsare the postpartum-derived cells described above. In another aspect, theinvention features a pharmaceutical composition comprising apharmaceutically acceptable carrier and a preparation made from thepostpartum cells, which may be a cell lysate (or fraction thereof) ofthe postpartum cells, an extracellular matrix of the postpartum cells ora conditioned medium in which the postpartum cells were grown. Kits forpracticing this aspect of the invention are also provided. These mayinclude the one or more of a pharmaceutically acceptable carrier orother agent or reagent, one or more of a cell lysate or fractionthereof, an extracellular matrix or a conditioned medium from thepostpartum cells, and instructions for use of the kit components.

Another aspect of the invention features a method for increasing thesurvival, growth or activity of cells for transplantation to treat anocular degenerative disorder. The method comprises co-culturing thecells for transplantation with cultured cells derived from postpartumplacental or umbilical tissue, under conditions effective to increasethe survival, growth or activity of the cells for transplantation.Preferably, the postpartum cells are the postpartum-derived cellsdescribed above. A kit for practicing the method is also provided. Thekit comprises the cultured postpartum cells and instructions forco-culturing the cells for transplantation with the postpartum cellsunder conditions effective to increase the survival, growth or activityof the cells for transplantation.

In one embodiment, the present invention provides a method for treatinga patient having a retinopathy or a retinal/macular disorder, the methodcomprising administering to the patient's eye umbilicus-derived cells,in an amount effective to treat the retinopathy or a retinal/maculardisorder, wherein the cells are capable of self-renewal and expansion inculture, have the potential to differentiate into cells of at least aneural phenotype, and have the following characteristics:

-   -   a. Potential for at least 40 population doublings in culture;    -   b. Attachment and expansion on a coated or uncoated tissue        culture vessel, wherein the coated tissue culture vessel        comprises a coating of gelatin, laminin, collagen,        polyornithine, vitronectin, or fibronectin;    -   c. Production of vimentin and alpha-smooth muscle actin;    -   d. Production of CD10, CD13, CD44, CD73, and CD90; and    -   e. Increased expression of genes encoding interleukin 8 and        reticulon 1 relative to a human cell that is a fibroblast, a        mesenchymal stem cell, or an iliac crest bone marrow cell.

In one embodiment, the umbilicus-derived cells are positive for HLA-A,B, C, and negative for CD31, CD34, CD45, CD117, and CD141. In oneembodiment, the umbilicus-derived cells are expanded in culture prior toadministering to the patient's eye.

In one embodiment, the retinopathy or a retinal/macular disorder isage-related macular degeneration. In an alternate embodiment, theretinopathy or a retinal/macular disorder is glaucoma.

In one embodiment, the present invention provides a method forpreventing the loss of photoreceptor cells associated with a retinopathyor a retinal/macular disorder in a patient, the method comprisingadministering to the patient's eye umbilicus-derived cells, in an amounteffective to prevent the loss of photoreceptor cells, wherein the cellsare capable of self-renewal and expansion in culture, have the potentialto differentiate into cells of at least a neural phenotype, and have thefollowing characteristics:

-   -   a. Potential for at least 40 population doublings in culture;    -   b. Attachment and expansion on a coated or uncoated tissue        culture vessel, wherein the coated tissue culture vessel        comprises a coating of gelatin, laminin, collagen,        polyornithine, vitronectin, or fibronectin;    -   c. Production of vimentin and alpha-smooth muscle actin;    -   d. Production of CD10, CD13, CD44, CD73, and CD90; and    -   e. Increased expression of genes encoding interleukin 8 and        reticulon 1 relative to a human cell that is a fibroblast, a        mesenchymal stem cell, or an iliac crest bone marrow cell.

In one embodiment, the umbilicus-derived cells are positive for HLA-A,B, C, and negative for CD31, CD34, CD45, CD117, and CD141. In oneembodiment, the umbilicus-derived cells are expanded in culture prior toadministering to the patient's eye.

In one embodiment, the retinopathy or a retinal/macular disorder isage-related macular degeneration. In an alternate embodiment, theretinopathy or a retinal/macular disorder is glaucoma.

In one embodiment, the loss of photoreceptor cells is prevented byinhibiting the apoptosis of the photoreceptor cells. In an alternateembodiment, the loss of photoreceptor cells is prevented by stimulatingthe phagocytosis of shed photoreceptor fragments.

In one embodiment, the umbilicus-derived cells phagocytose the shedphotoreceptor fragments. In an alternate embodiment, theumbilicus-derived cells stimulate the phagocytosis of shed photoreceptorfragments by RPE cells.

In one embodiment, the loss of photoreceptor cells is prevented byreplacing the photoreceptor cells. In one embodiment, the photoreceptorcells are replaced by the umbilicus-derived cells differentiatingretinal progenitor cells into photoreceptor cells.

In one embodiment, the loss of photoreceptor cells is prevented byre-surfacing Bruch's membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphical representation of the layout of the cells in atypical trans-well experiment.

FIG. 2 shows the effect of umbilicus-derived cells on phagocytosisobserved in aged primary human RPE cells.

FIG. 3 shows the analysis of human dermal fibroblast stimulatedphagocytosis.

FIG. 4 shows the effects of umbilicus-derived cells on the phagocytosisof fluorescein isothiocyanate—photoreceptor outer segments observed innormal and dystrophic RPE cells.

FIG. 5 shows the adherence and integration of umbilicus-derived cells onaged explants of human Bruch's membrane after 7 days.

FIG. 6 shows the phototransduction pathway in cone and rod cells.Shading denotes up-regulated expression.

FIG. 7 shows the dose-dependant effect of H₂O₂-induced DNA fragmentationin ARPE-19 cells. Filled square and solid line: normal growth media;open square and dotted line: media conditioned using umbilicus-derivedcells.

FIG. 8 shows the dose-dependant effect of H₂O₂-induced early apoptosisin ARPE-19 cells. Upper panel: early apoptotic cells: Annexin V(+) and7-ADD (−); lower panel: total apoptotic cells: Annexin V(+). Filledsquare and solid line: normal growth media; open square and dotted line:conditioned media.

FIG. 9 shows the time course of H₂O₂-induced DNA fragmentation inARPE-19 cells. Filled square and solid line: normal growth media; opensquare and dotted line: media conditioned using umbilicus-derived cells.Dotted line: media conditioned using c umbilicus-derived cells.

FIG. 10 shows a representative image of an injected eye. This eye wasembedded in paraffin and sectioned until the suture was visible on astereomicroscope and the image was collected. The eye shown was avehicle-injected eye 1 day post-injection. The suture placed followingthe injection procedure can be seen on the right side of the eye(arrow).

FIG. 11 shows the morphometric analysis of the outer nuclear layer(ONL). Shown is an eye 60 days post-injection, 2× magnification. Theregions of ONL analysis are outlined in boxes. Region 1 (bottom box) islocal to the injection site, and region 2 (top box) is distal to theinjection site, on the opposite side of the eye.

FIG. 12 shows the identification of umbilicus-derived cells insubretinal space, day 1 post-injection. Panel A: H&E stained section ofa day 1 eye at 20× magnification. Injected umbilicus-derived cells canbe seen in subretinal space. Panel B: an adjacent section immunostainedfor NuMA, 40× magnification. Infiltrating neutrophils can also beidentified amongst NuMA-positive umbilicus-derived cells.

FIG. 13 shows cell retention in the RCS rat eye. Cell retention in theRCS rat eyes from Day 1 to Day 60; the Y-axis is on logarithmic scale.Human β2M mRNA was detected in eyes 1, 7, 14, and 60 days aftersubretinal injection. The cell number reduced gradually from 18227±3227(mean+/−standard error of the mean; n=4) at Day 1 to 2266±1328(mean+/−standard error of the mean; n=4) at Day 60.

FIG. 14 shows the time course of ONL degeneration. H&E stained sectionsof control and umbilicus-derived cell injected eyes from the followingtime points post-injection: Day 7 (A and B), Day 14 (C and D), Day 30 (Eand F), and Day 60 (G and H). Images were acquired using 40×magnification near the injection site region. Arrows mark the outernuclear layer.

FIG. 15 demonstrates the injection of umbilicus-derived cells preservesONL thickness at Day 60 post-injection. Representative images from H&Estained sections from umbilicus-derived cell injected and control eyes60 days post-injection, 60× magnification. Panels A and B: Control eye,images taken from areas near and far from injection site, respectively.ONL (arrows) appears as a single discontinuous layer in both regions.Panels C and D: umbilicus-derived cell injected eye, areas near and farfrom injection site, respectively. ONL is visibly thicker in bothregions, although the area near the injection site is thicker than thatfurther away (approximately 4 nuclei thick near injection site comparedto 2 nuclei thick far from injection site in these images). Panels E andF: morphometric results. Panel E: All data from both regions analyzed(near to and far from injection site) were combined for each animal (n=3animals per group). Values graphed are the mean+/−standard deviation.Means were significantly different between control eyes and eyesinjected with umbilicus-derived cells (p<0.001, t-test). Panel F:Regional ONL rescue. The two columns on the left side of the graph arefrom region 1 (close to injection site), the two columns on the rightside are from region 2 (far from the injection site). Values graphed arethe mean+/−standard deviation of all tissue sections analyzed (forregion 1, the number of images were n=6 and 9 and region 2, the numberof images were n=9 and 6 for control and umbilicus-derived cells,respectively). The means for both groups were significantly different ineach region (p=0.002 using Mann-Whitney rank sum test for region 1, andp<0.001 using t-test for region 2. Mann-Whitney test was used for region1 because the umbilicus-derived cells group did not have a normaldistribution).

FIG. 16 demonstrates injection of umbilicus-derived cells preservesrhodopsin immunostaining at Day 60 post-injection. Panels A and B:representative images from Day 60 control and umbilicus-derived cellinjected animals at 60× magnification near the injection site. Panel A:control, panel B: umbilicus-derived cells. Rhodopsin immunostaining isspecific to the neuroepithelial layer (arrows) and is visibly increasedin the umbilicus-derived cell injected eye compared with control. PanelC: morphometric results, values graphed are the mean+/−standarddeviation (n=3 animals per group). Means were significantly differentbetween control eyes and eyes injected with umbilicus-derived cells:7.04+/−5.96 and 72.83+/−16.63, respectively (p=0.003, t-test). Valuesrepresent raw data multiplied by 100 to be expressed as a percentage.

FIG. 17 demonstrates injection of umbilicus-derived cells preservescalretinin immunostaining at Day 60 post-injection. Shown arerepresentative images from Day 60 control and umbilicus-derived cellinjected animals at 60× magnification near the injection site. Panel A:control, panel B: umbilicus-derived cells. Calretinin immunostaining isspecific to the inner nuclear layer (INL), inner plexiform layer (IPL),and the retinal ganglion cell layer (GCL). Panels C and D: morphometricresults, values graphed are the mean+/−standard deviation (n=3 animalsper group). Panel C: total calretinin staining (GCL, IPL, INL). Meanswere significantly different between control eyes and eyes injected withumbilicus-derived cells: 10.05+/−0.66 and 12.52+/−0.60, respectively(p=0.009, t-test). Panel D: calretinin staining of INL and GCL. Meanswere significantly different between control and umbilicus-derived cellinjected eyes in the INL but not in the GCL: 3.82+/−0.22 and 5.67+/−0.85(INL, p=0.014, t-test) and 2.91+/−1.11 and 3.16+/−0.37 (GCL, p=0.629,t-test), respectively.

FIG. 18 demonstrates injection of umbilicus-derived cells preservesrecoverin immunostaining at Day 60 post-injection. umbilicus-derivedcells significantly preserves recoverin immunostaining at day 60post-injection. Panels A and B: representative images from Day 60control and umbilicus-derived cell injected animals at 60× magnificationnear the injection site. Panel A: control, panel B: umbilicus-derivedcells. Recoverin immunostaining is specific to the outer nuclear layer(arrows) and a subpopulation of cells in the inner nuclear layer (arrowheads). Panels C and D: morphometric results, values graphed are themean+/−standard deviation (n=3 animals per group). Panel C: totalrecoverin staining (INL, ONL). Means were significantly differentbetween control eyes and eyes injected with umbilicus-derived cells:2.47+/−0.50 and 13.03+/−3.28, respectively (P=0.001, t-test). Panel D:recoverin staining of INL and ONL. Means were significantly differentbetween control and umbilicus-derived cell injected eyes in both the INLand ONL 1.11+/−0.31 and 2.24+/−0.51 (INL, p=0.031, t-test) and1.36+/−0.34 and 10.78+/−2.87 (ONL, p=0.001, t-test), respectively.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An embryonic stem cell is a pluripotent cell from the inner cellmass of a blastocyst-stage embryo. A fetal stem cell is one thatoriginates from fetal tissues or membranes. A postpartum stem cell is amultipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the placenta andthe umbilical cord. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiatedcell is one that has taken on a more specialized (“committed”) positionwithin the lineage of a cell. The term committed, when applied to theprocess of differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.De-differentiation refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e. which cells it came from and what cells it can give rise to. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into an ocular lineage orphenotype refers to a cell that becomes partially or fully committed toa specific ocular phenotype, including without limitation, retinal andcorneal stem cells, pigment epithelial cells of the retina and iris,photoreceptors, retinal ganglia and other optic neural lineages (e.g.,retinal glia, microglia, astrocytes, Mueller cells), cells forming thecrystalline lens, and epithelial cells of the sclera, cornea, limbus andconjunctiva. The phrase differentiates into a neural lineage orphenotype refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia.

The cells exemplified herein and preferred for use in present inventionare generally referred to as postpartum-derived cells (or PPDCs). Theyalso may sometimes be referred to more specifically as umbilicus-derivedcells or placenta-derived cells (UDCs or PDCs). In addition, the cellsmay be described as being stem or progenitor cells, the latter termbeing used in the broad sense. The term derived is used to indicate thatthe cells have been obtained from their biological source and grown invitro (e.g., cultured in a Growth Medium to expand the population and/orto produce a cell line). The in vitro manipulations of umbilical stemcells and the unique features of the umbilicus-derived cells of thepresent invention are described in detail below. Cells isolated frompostpartum placenta and umbilicus by other means is also consideredsuitable for use in the present invention. These other cells arereferred to herein as postpartum cells (rather than postpartum-derivedcells).

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture” or “cultured”). A primary cellculture is a culture of cells, tissues, or organs taken directly from anorganism(s) before the first subculture. Cells are expanded in culturewhen they are placed in a Growth Medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium,growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. When cells are cultured ina medium, they may secrete cellular factors that can provide trophicsupport to other cells. Such trophic factors include, but are notlimited to hormones, cytokines, extracellular matrix (ECM), proteins,vesicles, antibodies, and granules. The medium containing the cellularfactors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, differentiation, proliferation and/or maturation of acell, or stimulates increased activity of a cell. The interactionbetween cells via trophic factors may occur between cells of differenttypes. Cell interaction by way of trophic factors is found inessentially all cell types, and is a particularly significant means ofcommunication among neural cell types. Trophic factors also can functionin an autocrine fashion, i.e., a cell may produce trophic factors thataffect its own survival, growth, differentiation, proliferation and/ormaturation.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are alive and metabolically active, butthey do not divide. The nondividing state of senescent cells has not yetbeen found to be reversible by any biological, chemical, or viral agent.

The terms ocular, ophthalmic and optic are used interchangeably hereinto define “of, or about, or related to the eye.”

The term ocular degenerative condition (or disorder) is an inclusiveterm encompassing acute and chronic conditions, disorders or diseases ofthe eye, inclusive of the neural connection between the eye and thebrain, involving cell damage, degeneration or loss. An oculardegenerative condition may be age-related, or it may result from injuryor trauma, or it may be related to a specific disease or disorder. Acuteocular degenerative conditions include, but are not limited to,conditions associated with cell death or compromise affecting the eyeincluding conditions arising from cerebrovascular insufficiency, focalor diffuse brain trauma, diffuse brain damage, infection or inflammatoryconditions of the eye, retinal tearing or detachment, intra-ocularlesions (contusion penetration, compression, laceration) or otherphysical injury (e.g., physical or chemical burns). Chronic oculardegenerative conditions (including progressive conditions) include, butare not limited to, retinopathies and other retinal/macular disorderssuch as retinitis pigmentosa (RP), age-related macular degeneration(AMD), choroidal neovascular membrane (CNVM); retinopathies such asdiabetic retinopathy, occlusive retinopathy, sickle cell retinopathy andhypertensive retinopathy, central retinal vein occlusion, stenosis ofthe carotid artery, optic neuropathies such as glaucoma and relatedsyndromes; disorders of the lens and outer eye, e.g., limbal stem celldeficiency (LSCD), also referred to as limbal epithelial cell deficiency(LECD), such as occurs in chemical or thermal injury, Steven-Johnsonsyndrome, contact lens-induced keratopathy, ocular cicatricialpemphigoid, congenital diseases of aniridia or ectodermal dysplasia, andmultiple endocrine deficiency-associated keratitis.

The term treating (or treatment of) an ocular degenerative conditionrefers to ameliorating the effects of, or delaying, halting or reversingthe progress of, or delaying or preventing the onset of, an oculardegenerative condition as defined herein.

The term effective amount refers to a concentration or amount of areagent or pharmaceutical composition, such as a growth factor,differentiation agent, trophic factor, cell population or other agent,that is effective for producing an intended result, including cellgrowth and/or differentiation in vitro or in vivo, or treatment ofocular degenerative conditions, as described herein. With respect togrowth factors, an effective amount may range from about 1nanogram/milliliter to about 1 microgram/milliliter. With respect toPPDCs as administered to a patient in vivo, an effective amount mayrange from as few as several hundred or fewer, to as many as severalmillion or more. In specific embodiments, an effective amount may rangefrom 10³ to 10¹¹, more specifically at least about 10⁴ cells. It will beappreciated that the number of cells to be administered will varydepending on the specifics of the disorder to be treated, including butnot limited to size or total volume/surface area to be treated, as wellas proximity of the site of administration to the location of the regionto be treated, among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms that are not only compatible with the cells andother agents to be administered therapeutically, but also are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio.

Several terms are used herein with respect to cell replacement therapy.The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells and have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy. Transplantation as used herein refers to theintroduction of autologous, or allogeneic donor cell replacement therapyinto a recipient.

Description

Ocular degenerative conditions, which encompass acute, chronic andprogressive disorders and diseases having divergent causes, have as acommon feature the dysfunction or loss of a specific or vulnerable groupof ocular cells. This commonality enables development of similartherapeutic approaches for the repair or regeneration of vulnerable,damaged or lost ocular tissue, one of which is cell-based therapy.Development of cell therapy for ocular degenerative conditionsheretofore has been limited to a comparatively few types of stem orprogenitor cells, including ocular-derived stem cells themselves (e.g.,retinal and corneal stem cells), embryonic stem cells and a few types ofadult stem or progenitor cells (e.g., neural, mucosal epithelial andbone marrow stem cells). The present inventors have identified asignificant new source of stem cells for this purpose, namely, cellsisolated from the postpartum placenta and umbilical cord. Accordingly,in its various embodiments described herein, the present inventionfeatures methods and pharmaceutical compositions for repair andregeneration of ocular tissues, which utilize progenitor cells and cellpopulations isolated from postpartum tissues. The invention isapplicable to any ocular degenerative condition, but is expected to beparticularly suitable for a number of ocular disorders for whichtreatment or cure heretofore has been difficult or unavailable. Theseinclude, without limitation, age-related macular degeneration, retinitispigmentosa, diabetic and other retinopathies, glaucoma and other opticneuropathies, and disorders associated with limbal stem cell deficiency.

Stem or progenitor cells isolated from postpartum placenta or umbilicalcord in accordance with any method known in the art are expected to besuitable for use in the present invention. In a one embodiment, however,the invention utilizes postpartum-derived cells (PPDCs) as definedabove, which are derived from placental or umbilical cord tissue thathas been rendered substantially free of blood, preferably in accordancewith the method set forth below. The PPDCs are capable of self-renewaland expansion in culture and have the potential to differentiate intocells of other phenotypes. Certain embodiments features populationscomprising such cells, pharmaceutical compositions comprising the cellsor components or products thereof, and methods of using thepharmaceutical compositions for treatment of patients with acute orchronic ocular degenerative conditions. The postpartum-derived cells ofthe present invention have been characterized by their growth propertiesin culture, by their cell surface markers, by their gene expression, bytheir ability to produce certain biochemical trophic factors, and bytheir immunological properties.

Preparation of PPDCs

According to the methods described herein, a mammalian placenta andumbilical cord are recovered upon or shortly after termination of eithera full-term or pre-term pregnancy, for example, after expulsion afterbirth. The postpartum tissue may be transported from the birth site to alaboratory in a sterile container such as a flask, beaker, culture dish,or bag. The container may have a solution or medium, including but notlimited to a salt solution, such as, for example, Dulbecco's ModifiedEagle's Medium (DMEM) or phosphate buffered saline (PBS), or anysolution used for transportation of organs used for transplantation,such as University of Wisconsin solution or perfluorochemical solution.One or more antibiotic and/or antimycotic agents, such as but notlimited to penicillin, streptomycin, amphotericin B, gentamicin, andnystatin, may be added to the medium or buffer. The postpartum tissuemay be rinsed with an anticoagulant solution such as heparin-containingsolution. It is preferable to keep the tissue at about 4-10° C. prior toextraction of PPDCs. It is even more preferable that the tissue not befrozen prior to extraction of PPDCs.

Isolation of PPDCs preferably occurs in an aseptic environment. Theumbilical cord may be separated from the placenta by means known in theart. Alternatively, the umbilical cord and placenta are used withoutseparation. Blood and debris are preferably removed from the postpartumtissue prior to isolation of PPDCs. For example, the postpartum tissuemay be washed with buffer solution, such as but not limited to phosphatebuffered saline. The wash buffer also may comprise one or moreantimycotic and/or antibiotic agents, such as but not limited topenicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Postpartum tissue comprising a whole placenta or a fragment or sectionthereof is disaggregated by mechanical force (mincing or shear forces).In a presently preferred embodiment, the isolation procedure alsoutilizes an enzymatic digestion process. Many enzymes are known in theart to be useful for the isolation of individual cells from complextissue matrices to facilitate growth in culture. Ranging from weaklydigestive (e.g. deoxyribonucleases and the neutral protease, dispase) tostrongly digestive (e.g. papain and trypsin), such enzymes are availablecommercially. A nonexhaustive list of enzymes compatible herewithincludes mucolytic enzyme activities, metalloproteases, neutralproteases, serine proteases (such as trypsin, chymotrypsin, orelastase), and deoxyribonucleases. Presently preferred are enzymeactivities selected from metalloproteases, neutral proteases andmucolytic activities. For example, collagenases are known to be usefulfor isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell clumping during isolation.Preferred methods involve enzymatic treatment with for examplecollagenase and dispase, or collagenase, dispase, and hyaluronidase, andsuch methods are provided wherein in certain preferred embodiments, amixture of collagenase and the neutral protease dispase are used in thedissociating step. More preferred are those methods that employdigestion in the presence of at least one collagenase from Clostridiumhistolyticum, and either of the protease activities, dispase andthermolysin. Still more preferred are methods employing digestion withboth collagenase and dispase enzyme activities. Also preferred aremethods that include digestion with a hyaluronidase activity in additionto collagenase and dispase activities. The skilled artisan willappreciate that many such enzyme treatments are known in the art forisolating cells from various tissue sources. For example, the LIBERASEBlendzyme (Roche) series of enzyme combinations are suitable for use inthe instant methods. Other sources of enzymes are known, and the skilledartisan may also obtain such enzymes directly from their naturalsources. The skilled artisan is also well equipped to assess new, oradditional enzymes or enzyme combinations for their utility in isolatingthe cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5,or 2 hours long or longer. In other preferred embodiments, the tissue isincubated at 37° C. during the enzyme treatment of the dissociationstep.

In some embodiments of the invention, postpartum tissue is separatedinto sections comprising various aspects of the tissue, such asneonatal, neonatal/maternal, and maternal aspects of the placenta, forinstance. The separated sections then are dissociated by mechanicaland/or enzymatic dissociation according to the methods described herein.Cells of neonatal or maternal lineage may be identified by any meansknown in the art, for example, by karyotype analysis or in situhybridization for a Y chromosome.

Isolated cells or postpartum tissue from which PPDCs grow out may beused to initiate, or seed, cell cultures. Isolated cells are transferredto sterile tissue culture vessels either uncoated or coated withextracellular matrix or ligands such as laminin, collagen (native,denatured or crosslinked), gelatin, fibronectin, and other extracellularmatrix proteins. PPDCs are cultured in any culture medium capable ofsustaining growth of the cells such as, but not limited to, DMEM (highor low glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium,Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modifiedDulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),DMEM/F12, RPMI 1640, and cellgro® FREE™. The culture medium may besupplemented with one or more components including, for example, fetalbovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES);human serum (HS); beta-mercaptoethanol (BME or 2-ME), preferably about0.001% (v/v); one or more growth factors, for example, platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) anderythropoietin; amino acids, including L-valine; and one or moreantibiotic and/or antimycotic agents to control microbial contamination,such as, for example, penicillin G, streptomycin sulfate, amphotericinB, gentamicin, and nystatin, either alone or in combination. The culturemedium preferably comprises Growth Medium (DMEM-low glucose, serum, BME,and an antibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. In a preferred embodiment, the cells are cultured at about 0 toabout 5 percent by volume CO₂ in air. In some preferred embodiments, thecells are cultured at about 2 to about 25 percent O₂ in air, preferablyabout 5 to about 20 percent O₂ in air. The cells preferably are culturedat about 25 to about 40° C. and more preferably are cultured at 37° C.The cells are preferably cultured in an incubator. The medium in theculture vessel can be static or agitated, for example, using abioreactor. PPDCs preferably are grown under low oxidative stress (e.g.,with addition of glutathione, Vitamin C, Catalase, Vitamin E,N-Acetylcysteine). “Low oxidative stress”, as used herein, refers toconditions of no or minimal free radical damage to the cultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

After culturing the isolated cells or tissue fragments for a sufficientperiod of time, PPDCs will have grown out, either as a result ofmigration from the postpartum tissue or cell division, or both. In someembodiments of the invention, PPDCs are passaged, or removed to aseparate culture vessel containing fresh medium of the same or adifferent type as that used initially, where the population of cells canbe mitotically expanded. The cells of the invention may be used at anypoint between passage 0 and senescence. The cells preferably arepassaged between about 3 and about 25 times, more preferably arepassaged about 4 to about 12 times, and preferably are passaged 10 or 11times. Cloning and/or subcloning may be performed to confirm that aclonal population of cells has been isolated.

In some aspects of the invention, the different cell types present inpostpartum tissue are fractionated into subpopulations from which thePPDCs can be isolated. This may be accomplished using standardtechniques for cell separation including, but not limited to, enzymatictreatment to dissociate postpartum tissue into its component cells,followed by cloning and selection of specific cell types, for examplebut not limited to selection based on morphological and/or biochemicalmarkers; selective growth of desired cells (positive selection),selective destruction of unwanted cells (negative selection); separationbased upon differential cell agglutinability in the mixed population as,for example, with soybean agglutinin; freeze-thaw procedures;differential adherence properties of the cells in the mixed population;filtration; conventional and zonal centrifugation; centrifugalelutriation (counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and fluorescence activatedcell sorting (FACS). For a review of clonal selection and cellseparation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: AMANUAL OF BASIC TECHNIQUES, 3rd Ed., Wiley-Liss, Inc., New York, whichis incorporated herein by reference.

The culture medium is changed as necessary, for example, by carefullyaspirating the medium from the dish, for example, with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulates in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to bePPDCs.

PPDCs may be cryopreserved. Accordingly, in a preferred embodimentdescribed in greater detail below, PPDCs for autologous transfer (foreither the mother or child) may be derived from appropriate postpartumtissues following the birth of a child, then cryopreserved so as to beavailable in the event they are later needed for transplantation.

Characteristics of PPDCs

PPDCs may be characterized, for example, by growth characteristics(e.g., population doubling capability, doubling time, passages tosenescence), karyotype analysis (e.g., normal karyotype; maternal orneonatal lineage), flow cytometry (e.g., FACS analysis),immunohistochemistry and/or immunocytochemistry (e.g., for detection ofepitopes), gene expression profiling (e.g., gene chip arrays; polymerasechain reaction (for example, reverse transcriptase PCR, real time PCR,and conventional PCR)), protein arrays, protein secretion (e.g., byplasma clotting assay or analysis of PDC-conditioned medium, forexample, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocytereaction (e.g., as measure of stimulation of PBMCs), and/or othermethods known in the art.

Examples of PPDCs derived from placental tissue were deposited with theAmerican Type Culture Collection (ATCC, Manassas, Va.) and assigned ATCCAccession Numbers as follows: (1) strain designation PLA 071003 (P8) wasdeposited Jun. 15, 2004 and assigned Accession No. PTA-6074; (2) straindesignation PLA 071003 (P11) was deposited Jun. 15, 2004 and assignedAccession No. PTA-6075; and (3) strain designation PLA 071003 (P16) wasdeposited Jun. 16, 2004 and assigned Accession No. PTA-6079. Examples ofPPDCs derived from umbilicus tissue were deposited with the AmericanType Culture Collection on Jun. 10, 2004, and assigned ATCC AccessionNumbers as follows: (1) strain designation UMB 022803 (P7) was assignedAccession No. PTA-6067; and (2) strain designation UMB 022803 (P17) wasassigned Accession No. PTA-6068.

In various embodiments, the PPDCs possess one or more of the followinggrowth features (1) they require L-valine for growth in culture; (2)they are capable of growth in atmospheres containing oxygen from about5% to at least about 20% (3) they have the potential for at least about40 doublings in culture before reaching senescence; and (4) they attachand expand on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyomithine, vitronectin or fibronectin.

In certain embodiments the PPDCs possess a normal karyotype, which ismaintained as the cells are passaged. Karyotyping is particularly usefulfor identifying and distinguishing neonatal from maternal cells derivedfrom placenta. Methods for karyotyping are available and known to thoseof skill in the art.

In other embodiements, the PPDCs may be characterized by production ofcertain proteins, including (1) production of at least one of tissuefactor, vimentin, and alpha-smooth muscle actin; and (2) production ofat least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 andHLA-A,B,C cell surface markers, as detected by flow cytometry. In otherembodiments, the PPDCs may be characterized by lack of production of atleast one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2,HLA-G, and HLA-DR,DP,DQ cell surface markers, as detected by flowcytometry. Paryicularly preferred are cells that produce at least two oftissue factor, vimentin, and alpha-smooth muscle actin. More preferredare those cells producing all three of the proteins tissue factor,vimentin, and alpha-smooth muscle actin.

In other embodiments, the PPDCs may be characterized by gene expression,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an iliac crest bone marrow cell, is increased for a geneencoding at least one of interleukin 8; reticulon 1; chemokine (C—X—Cmotif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine(C—X—C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C—X—C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3;C-type lectin superfamily member 2; Wilms tumor 1; aldehydedehydrogenase 1 family member A2; renin; oxidized low densitylipoprotein receptor 1; Homo sapiens clone IMAGE:4179671; protein kinaseC zeta; hypothetical protein DKFZp564F013; downregulated in ovariancancer 1; and Homo sapiens gene from clone DKFZp547k1113.

In yet other embodiments, the PPDCs may be characterized by geneexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is reducedfor a gene encoding at least one of: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C—X—C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeo box 2(growth arrest-specifichomeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAAI034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein FLJ20373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (T AZ); fibronectin 1; proenkephalin; integrin,beta-like 1(with EGF-like repeat domains); Homo sapiens mRNA full lengthinsert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E IB 19 kDainteracting protein 3-like; AE binding protein 1; and cytochrome coxidase subunit V ila polypeptide 1 (muscle).

In other embodiments, the PPDCs may be characterized by secretion of atleast one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO,MIP1a, RANTES, and TIMP1. In alternative embodiments, the PPDCs may becharacterized by lack of secretion of at least one of TGF-beta2, ANG2,PDGFbb, MIP1b, 1309, MDC, and VEGF, as detected by ELISA.

In preferred embodiments, the cell comprises two or more of theabove-listed growth, protein/surface marker production, gene expressionor substance-secretion characteristics. More preferred are those cellscomprising, three, four, or five or more of the characteristics. Stillmore preferred are PPDCs comprising six, seven, or eight or more of thecharacteristics. Still more preferred presently are those cellscomprising all of above characteristics.

Among cells that are presently preferred for use with the invention inseveral of its aspects are postpartum cells having the characteristicsdescribed above and more particularly those wherein the cells havenormal karyotypes and maintain normal karyotypes with passaging, andfurther wherein the cells express each of the markers CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, and HLA-A, B, C, wherein the cells produce theimmunologically-detectable proteins which correspond to the listedmarkers. Still more preferred are those cells which in addition to theforegoing do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD117, CD141, or HLA-DR, DP, DQ, as detected by flowcytometry.

Certain cells having the potential to differentiate along lines leadingto various phenotypes are unstable and thus can spontaneouslydifferentiate. Presently preferred for use with the invention are cellsthat do not spontaneously differentiate, for example along neural lines.Preferred cells, when grown in Growth Medium, are substantially stablewith respect to the cell markers produced on their surface, and withrespect to the expression pattern of various genes, for example asdetermined using an Affymetrix GENECHIP. The cells remain substantiallyconstant, for example in their surface marker characteristics overpassaging, through multiple population doublings.

However, one feature of PPDCs is that they may be deliberately inducedto differentiate into various lineage phenotypes by subjecting them todifferentiation-inducing cell culture conditions. Of use in treatment ofcertain ocular degenerative conditions, the PPDCs may be induced todifferentiate into neural phenotypes using one or more methods known inthe art. For instance, as exemplified herein, PPDCs may be plated onflasks coated with laminin in Neurobasal-A medium (Invitrogen, Carlsbad,Calif.) containing B27 (B27 supplement, Invitrogen), L-glutamine andPenicillin/Streptomycin, the combination of which is referred to hereinas Neural Progenitor Expansion (NPE) medium. NPE media may be furthersupplemented with bFGF and/or EGF. Alternatively, PPDCs may be inducedto differentiate in vitro by (1) co-culturing the PPDCs with neuralprogenitor cells, or (2) growing the PPDCs in neural progenitorcell-conditioned medium.

Differentiation of the PPDCs into neural phenotypes may be demonstratedby a bipolar cell morphology with extended processes. The induced cellpopulations may stain positive for the presence of nestin.Differentiated PPDCs may be assessed by detection of nestin, TuJ1 (BIIItubulin), GFAP, tyrosine hydroxylase, GABA, 04 and/or MBP. In someembodiments, PPDCs have exhibited the ability to form three-dimensionalbodies characteristic of neuronal stem cell formation of neurospheres.

Cell Populations, Modifications, Components and Products

Another aspect of the invention features populations of cells isolatedfrom placental or umbilical tissue. In a preferred embodiment, thepopulations comprise the PPDCs described above, and these cellpopulations are described in the section below. In some embodiments, thecell population is heterogeneous. A heterogeneous cell population of theinvention may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% PPDCs. The heterogeneous cell populations of theinvention may further comprise stem cells or other progenitor cells,such as epithelial or neural progenitor cells, or it may furthercomprise fully differentiated cells. In some embodiments, the populationis substantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). Thehomogeneous cell population of the invention may comprise umbilicus- orplacenta-derived cells. Homogeneous populations of umbilicus-derivedcells are preferably free of cells of maternal lineage. Homogeneouspopulations of placenta-derived cells may be of neonatal or maternallineage. Homogeneity of a cell population may be achieved by any methodknown in the art, for example, by cell sorting (e.g., flow cytometry) orby clonal expansion in accordance with known methods. Thus, preferredhomogeneous PPDC populations may comprise a clonal cell line ofpostpartum-derived cells. Such populations are particularly useful whena cell clone with highly desirable functionality has been isolated.

Also provided herein are populations of cells incubated in the presenceof one or more factors, or under conditions, that stimulate stem celldifferentiation along a desired pathway (e.g., neural, epithelial). Suchfactors are known in the art and the skilled artisan will appreciatethat determination of suitable conditions for differentiation can beaccomplished with routine experimentation. Optimization of suchconditions can be accomplished by statistical experimental design andanalysis, for example response surface methodology allows simultaneousoptimization of multiple variables, for example in a biological culture.Presently preferred factors include, but are not limited to factors,such as growth or trophic factors, demethylating agents, co-culture withneural or epithelial lineage cells or culture in neural or epitheliallineage cell-conditioned medium, as well other conditions known in theart to stimulate stem cell differentiation along these pathways (forfactors useful in neural differentiation, see, e.g., Lang, K. J. D. etal., 2004, J. Neurosci. Res. 76: 184-192; Johe, K. K. et al., 1996,Genes Devel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25:381-407).

Postpartum cells, preferably PPDCs, may also be genetically modified toproduce therapeutically useful gene products, or to produceantineoplastic agents for treatment of tumors, for example. Geneticmodification may be accomplished using any of a variety of vectorsincluding, but not limited to, integrating viral vectors, e.g.,retrovirus vector or adeno-associated viral vectors; non-integratingreplicating vectors, e.g., papilloma virus vectors, SV40 vectors,adenoviral vectors; or replication-defective viral vectors. Othermethods of introducing DNA into cells include the use of liposomes,electroporation, a particle gun, or by direct DNA injection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by or in operative association with, one or more appropriateexpression control elements such as promoter or enhancer sequences,transcription terminators, polyadenylation sites, among others, and aselectable marker. Any promoter may be used to drive the expression ofthe inserted gene. For example, viral promoters include, but are notlimited to, the CMV promoter/enhancer, SV 40, papillomavirus,Epstein-Barr virus or elastin gene promoter. In some embodiments, thecontrol elements used to control expression of the gene of interest canallow for the regulated expression of the gene so that the product issynthesized only when needed in vivo. If transient expression isdesired, constitutive promoters are preferably used in a non-integratingand/or replication-defective vector. Alternatively, inducible promoterscould be used to drive the expression of the inserted gene whennecessary. Inducible promoters include, but are not limited to thoseassociated with metallothionein and heat shock proteins.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines. This methodcan be advantageously used to engineer cell lines that express the geneproduct.

Cells may be genetically engineered to “knock out” or “knock down”expression of factors that promote inflammation or rejection at theimplant site. Negative modulatory techniques for the reduction of targetgene expression levels or target gene product activity levels arediscussed below. “Negative modulation,” as used herein, refers to areduction in the level and/or activity of target gene product relativeto the level and/or activity of the target gene product in the absenceof the modulatory treatment. The expression of a gene native to a neuronor glial cell can be reduced or knocked out using a number of techniquesincluding, for example, inhibition of expression by inactivating thegene using the homologous recombination technique. Typically, an exonencoding an important region of the protein (or an ex on 5′ to thatregion) is interrupted by a positive selectable marker, e.g., neo,preventing the production of normal mRNA from the target gene andresulting in inactivation of the gene. A gene may also be inactivated bycreating a deletion in part of a gene, or by deleting the entire gene.By using a construct with two regions of homology to the target genethat are far apart in the genome, the sequences intervening the tworegions can be deleted (Mombaerts et al., 1991, Proc. Nat. Acad. Sci.U.S.A. 88:3084-3087). Antisense, DNAzymes, ribozymes, small interferingRNA (siRNA) and other such molecules that inhibit expression of thetarget gene can also be used to reduce the level of target geneactivity. For example, antisense RNA molecules that inhibit theexpression of major histocompatibility gene complexes (HLA) have beenshown to be most versatile with respect to immune responses. Stillfurther, triple helix molecules can be utilized in reducing the level oftarget gene activity. These techniques are described in detail by L. G.Davis et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY, 2nd ed.,Appleton & Lange, Norwalk, CN.

In other aspects, the invention provides cell lysates and cell solublefractions prepared from postpartum stem cells, preferably PPDCs, orheterogeneous or homogeneous cell populations comprising PPDCs, as wellas PPDCs or populations thereof that have been genetically modified orthat have been stimulated to differentiate along a neurogenic pathway.Such lysates and fractions thereof have many utilities. Use of the celllysate soluble fraction (i.e., substantially free of membranes) in vivo,for example, allows the beneficial intracellular milieu to be usedallogeneically in a patient without introducing an appreciable amount ofthe cell surface proteins most likely to trigger rejection, or otheradverse immunological responses. Methods of lysing cells are well knownin the art and include various means of mechanical disruption, enzymaticdisruption, or chemical disruption, or combinations thereof. Such celllysates may be prepared from cells directly in their Growth Medium andthus containing secreted growth factors and the like, or may be preparedfrom cells washed free of medium in, for example, PBS or other solution.Washed cells may be resuspended at concentrations greater than theoriginal population density if preferred.

In one embodiment, whole cell lysates are prepared, e.g., by disruptingcells without subsequent separation of cell fractions. In anotherembodiment, a cell membrane fraction is separated from a solublefraction of the cells by routine methods known in the art, e.g.,centrifugation, filtration, or similar methods.

Cell lysates or cell soluble fractions prepared from populations ofpostpartum-derived cells may be used as is, further concentrated, by forexample, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically-acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Cell lysates or fractions thereof may be used in vitro or in vivo, aloneor for example, with autologous or syngeneic live cells. The lysates, ifintroduced in vivo, may be introduced locally at a site of treatment, orremotely to provide, for example needed cellular growth factors to apatient.

In a further embodiment, postpartum cells, preferably PPDCs, can becultured in vitro to produce biological products in high yield. Forexample, such cells, which either naturally produce a particularbiological product of interest (e.g., a trophic factor), or have beengenetically engineered to produce a biological product, can be clonallyexpanded using the culture techniques described herein. Alternatively,cells may be expanded in a medium that induces differentiation to adesired lineage. In either case, biological products produced by thecell and secreted into the medium can be readily isolated from theconditioned medium using standard separation techniques, e.g., such asdifferential protein precipitation, ion-exchange chromatography, gelfiltration chromatography, electrophoresis, and HPLC, to name a few. A“bioreactor” may be used to take advantage of the flow method forfeeding, for example, a three-dimensional culture in vitro. Essentially,as fresh media is passed through the three-dimensional culture, thebiological product is washed out of the culture and may then be isolatedfrom the outflow, as above.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells be lysed, asdescribed above. The biological product may then be purified using anyone or more of the above-listed techniques.

In other embodiments, the invention provides conditioned medium fromcultured postpartum cells, preferably PPDCs, for use in vitro and invivo as described below. Use of such conditioned medium allows thebeneficial trophic factors secreted by the postpartum cells to be usedallogeneically in a patient without introducing intact cells that couldtrigger rejection, or other adverse immunological responses. Conditionedmedium is prepared by culturing cells in a culture medium, then removingthe cells from the medium.

Conditioned medium prepared from populations of postpartum-derived cellsmay be used as is, further concentrated, by for example, ultrafiltrationor lyophilization, or even dried, partially purified, combined withpharmaceutically-acceptable carriers or diluents as are known in theart, or combined with other compounds such as biologicals, for examplepharmaceutically useful protein compositions. Conditioned medium may beused in vitro or in vivo, alone or for example, with autologous orsyngeneic live cells. The conditioned medium, if introduced in vivo, maybe introduced locally at a site of treatment, or remotely to provide,for example needed cellular growth or trophic factors to a patient.

In another embodiment, an extracellular matrix (ECM) produced byculturing postpartum cells (preferably PPDCs) on liquid, solid orsemi-solid substrates is prepared, collected and utilized as analternative to implanting live cells into a subject in need of tissuerepair or replacement. The cells are cultured in vitro, on a threedimensional framework as described elsewhere herein, under conditionssuch that a desired amount of ECM is secreted onto the framework. Thecells and the framework are removed, and the ECM processed for furtheruse, for example, as an injectable preparation. To accomplish this,cells on the framework are killed and any cellular debris removed fromthe framework. This process may be carried out in a number of differentways. For example, the living tissue can be flash-frozen in liquidnitrogen without a cryopreservative, or the tissue can be immersed insterile distilled water so that the cells burst in response to osmoticpressure.

Once the cells have been killed, the cellular membranes may be disruptedand cellular debris removed by treatment with a mild detergent rinse,such as EDTA, CHAPS or a zwitterionic detergent. Alternatively, thetissue can be enzymatically digested and/or extracted with reagents thatbreak down cellular membranes and allow removal of cell contents.Example of such enzymes include, but are not limited to, hyaluronidase,dispase, proteases, and nucleases. Examples of detergents includenon-ionic detergents such as, for example, alkylaryl polyether alcohol(TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and HaasPhiladelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl ether (AtlasChemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), apolyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylenelauryl ether (Rohm and Haas); and ionic detergents such as, for example,sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonatedalkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in abranched or unbranched chain.

The collection of the ECM can be accomplished in a variety of ways,depending, for example, on whether the new tissue has been formed on athree-dimensional framework that is biodegradable or non-biodegradable.For example, if the framework is non-biodegradable, the ECM can beremoved by subjecting the framework to sonication, high-pressure waterjets, mechanical scraping, or mild treatment with detergents or enzymes,or any combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM.

After it has been collected, the ECM may be processed further. Forexample, the ECM can be homogenized to fine particles using techniqueswell known in the art such as by sonication, so that it can pass througha surgical needle. The components of the ECM can be crosslinked, ifdesired, by gamma irradiation. Preferably, the ECM can be irradiatedbetween 0.25 to 2 mega rads to sterilize and cross link the ECM.Chemical crosslinking using agents that are toxic, such asglutaraldehyde, is possible but not generally preferred.

The amounts and/or ratios of proteins, such as the various types ofcollagen present in the ECM, may be adjusted by mixing the ECM producedby the cells of the invention with ECM of one or more other cell types.In addition, biologically active substances such as proteins, growthfactors and/or drugs, can be incorporated into the ECM. Exemplarybiologically active substances include tissue growth factors, such asTGF-beta, and the like, which promote healing and tissue repair at thesite of the injection. Such additional agents may be utilized in any ofthe embodiments described herein above, e.g., with whole cell lysates,soluble cell fractions, or further purified components and productsproduced by the cells.

In another embodiment, postpartum cells (preferably PPDCs) are used tore-surface Bruch's membrane that has undergone modifications due to age.Such modifications include, for example, increased thickness, depositionof extracellular matrix and lipids, cross-linking of protein,non-enzymatic formation of advanced glycation end products. In oneembodiment, the age-related changes in Bruch's membrane causedissociation of RPE cells from the Bruch's membrane, and ultimatelyresult in photoreceptor cell death. In one aspect of the presentinvention, the resurfacing of Bruch's membrane by postpartum cells(preferably PPDCs) prevents the dissociation of RPE cells from theBruch's membrane. In an alternate embodiment, the postpartum cellspromote the attachment of RPE cells to Bruch's membrane. The RPE cellsmay be endogenous, or they may be transplanted RPE cells.

Pharmaceutical Compositions

In another aspect, the invention provides pharmaceutical compositionsthat utilize postpartum cells, preferably PPDCs, cell populations andcell components and products in various methods for treatment of oculardegenerative conditions. Certain embodiments encompass pharmaceuticalcompositions comprising live cells (e.g., PPDCs alone or admixed withother cell types). Other embodiments encompass pharmaceuticalcompositions comprising PPDC cellular components (e.g., cell lysates,soluble cell fractions, conditioned medium, ECM, or components of any ofthe foregoing) or products (e.g., trophic and other biological factorsproduced naturally by PPDCs or through genetic modification, conditionedmedium from PPDC culture). In either case, the pharmaceuticalcomposition may further comprise other active agents, such asanti-inflammatory agents, anti-apoptotic agents, antioxidants, growthfactors, neurotrophic factors or neuroregenerative, neuroprotective orophthalmic drugs as known in the art.

Examples of other components that may be added to PPDC pharmaceuticalcompositions include, but are not limited to: (1) other neuroprotectiveor neurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, PPDCs may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics,to name a few.

Pharmaceutical compositions of the invention comprise postpartum cells(preferably PPDCs), or components or products thereof, formulated with apharmaceutically acceptable carrier or medium. Suitable pharmaceuticallyacceptable carriers include water, salt solution (such as Ringer'ssolution), alcohols, oils, gelatins, and carbohydrates, such as lactose,amylose, or starch, fatty acid esters, hydroxymethylcellulose, andpolyvinyl pyrrolidine. Such preparations can be sterilized, and ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, and coloring. Typically, but not exclusively,pharmaceutical compositions comprising cellular components or products,but not live cells, are formulated as liquids. Pharmaceuticalcompositions comprising PPDC live cells are typically formulated asliquids, semisolids (e.g., gels) or solids (e.g., matrices, scaffoldsand the like, as appropriate for ophthalmic tissue engineering).

Pharmaceutical compositions may comprise auxiliary components as wouldbe familiar to medicinal chemists or biologists. For example, they maycontain antioxidants in ranges that vary depending on the kind ofantioxidant used. Reasonable ranges for commonly used antioxidants areabout 0.01% to about 0.15% weight by volume of EDTA, about 0.01% toabout 2.0% weight volume of sodium sulfite, and about 0.01% to about2.0% weight by volume of sodium metabisulfite. One skilled in the artmay use a concentration of about 0.1% weight by volume for each of theabove. Other representative compounds include mercaptopropionyl glycine,N-acetyl cysteine, beta-mercaptoethylamine, glutathione and similarspecies, although other anti-oxidant agents suitable for ocularadministration, e.g. ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent may be used to maintain the pH of eye dropformulations in the range of about 4.0 to about 8.0; so as to minimizeirritation of the eye. For direct intravitreal or intraocular injection,formulations should be at pH 7.2 to 7.5, preferably at pH 7.3-7.4. Theopthalmologic compositions may also include tonicity agents suitable foradministration to the eye. Among those suitable is sodium chloride tomake formulations approximately isotonic with 0.9% saline solution.

In certain embodiments, pharmaceutical compositions are formulated withviscosity enhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Thepharmaceutical compositions may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives mayalso be included, e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methylor propylparabens.

Formulations for injection are preferably designed for single-useadministration and do not contain preservatives. Injectable solutionsshould have isotonicity equivalent to 0.9% sodium chloride solution(osmolality of 290-300 milliosmoles). This may be attained by additionof sodium chloride or other co-solvents as listed above, or excipientssuch as buffering agents and antioxidants, as listed above.

The tissues of the anterior chamber of the eye are bathed by the aqueoushumor, while the retina is under continuous exposure to the vitreous.These fluids/gels exist in a highly reducing redox state because theycontain antioxidant compounds and enzymes. Therefore, it may beadvantageous to include a reducing agent in the opthalmologiccompositions. Suitable reducing agents include N-acetylcysteine,ascorbic acid or a salt form, and sodium sulfite or metabisulfite, withascorbic acid and/or N-acetylcysteine or glutathione being particularlysuitable for injectable solutions.

Pharmaceutical compositions comprising cells, cell components or cellproducts may be delivered to the eye of a patient in one or more ofseveral delivery modes known in the art. In one embodiment that may besuitable for use in some instances, the compositions are topicallydelivered to the eye in eye drops or washes. In another embodiment, thecompositions may be delivered to various locations within the eye viaperiodic intraocular injection or by infusion in an irrigating solutionsuch as BSS or BSS PLUS (Alcon USA, Fort Worth, Tex.). Alternatively,the compositions may be applied in other opthalmologic dosage formsknown to those skilled in the art, such as pre-formed or in situ-formedgels or liposomes, for example as disclosed in U.S. Pat. No. 5,718,922to Herrero-Vanrell. In another embodiment, the composition may bedelivered to or through the lens of an eye in need of treatment via acontact lens (e.g. Lidofilcon B, Bausch & Lomb CW79 or DELTACON(Deltafilcon A) or other object temporarily resident upon the surface ofthe eye. In other embodiments, supports such as a collagen cornealshield (e.g. BIO-COR dissolvable corneal shields, Summit Technology,Watertown, Mass.) can be employed. The compositions can also beadministered by infusion into the eyeball, either through a cannula froman osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) or byimplantation of timed-release capsules (OCCUSENT) or biodegradable disks(OCULEX, OCUSERT). These routes of administration have the advantage ofproviding a continuous supply of the pharmaceutical composition to theeye. This may be an advantage for local delivery to the cornea, forexample.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are typically formulated for surgical implantation at thesite of ocular damage or distress. It will be appreciated that liquidcompositions also may be administered by surgical procedures. Inparticular embodiments, semi-solid or solid pharmaceutical compositionsmay comprise semi-permeable gels, lattices, cellular scaffolds and thelike, which may be non-biodegradable or biodegradable. For example, incertain embodiments, it may be desirable or appropriate to sequester theexogenous cells from their surroundings, yet enable the cells to secreteand deliver biological molecules to surrounding cells. In theseembodiments, cells may be formulated as autonomous implants comprisingliving PPDCs or cell population comprising PPDCs surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression (for a reviewof such devices and methods, see, e.g., P. A. Tresco et al., 2000, Adv.Drug Delivery Rev. 42: 3-27).

In other embodiments, different varieties of degradable gels andnetworks are utilized for the pharmaceutical compositions of theinvention. For example, degradable materials particularly suitable forsustained release formulations include biocompatible polymers, such aspoly (lactic acid), poly (lactic-co-glycolic acid), methylcellulose,hyaluronic acid, collagen, and the like. The structure, selection anduse of degradable polymers in drug delivery vehicles have been reviewedin several publications, including, A. Domb et al., 1992, Polymers forAdvanced Technologies 3:279. U.S. Pat. No. 5,869,079 to Wong et al.disclose combinations of hydrophilic and hydrophobic entities in abiodegradable sustained release ocular implant. In addition, U.S. Pat.No. 6,375,972 to Guo et al., U.S. Pat. No. 5,902,598 to Chen et al.,U.S. Pat. No. 6,331,313 to Wong et al., U.S. Pat. No. 5,707,643 to Oguraet al., U.S. Pat. No. 5,466,233 to Weiner et al. and U.S. Pat. No.6,251,090 to Avery et al. each describes intraocular implant devices andsystems that may be used to deliver pharmaceutical compositions.

In other embodiments, e.g., for repair of neural lesions, such as adamaged or severed optic nerve, it may be desirable or appropriate todeliver the cells on or in a biodegradable, preferably bioresorbable orbioabsorbable, scaffold or matrix. These typically three-dimensionalbiomaterials contain the living cells attached to the scaffold,dispersed within the scaffold, or incorporated in an extracellularmatrix entrapped in the scaffold. Once implanted into the target regionof the body, these implants become integrated with the host tissue,wherein the transplanted cells gradually become established (see, e.g.,P. A. Tresco et al., 2000, supra; see also D. W. Hutmacher, 2001, J.Biomater. Sci. Polymer Edn. 12: 107-174).

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present invention includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe trade name VICRYL (Ethicon, Inc., Somerville, N.J.), Foams, composedof, for example, poly (epsilon-caprolactone)/poly (glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699 also may beutilized. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. In situ-forming degradable networks are also suitable foruse in the invention (see, e.g., Anseth, K. S. et al., 2002, J.Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24:3969-3980; U.S. Patent Publication 2002/0022676 to He et al.). Thesematerials are formulated as fluids suitable for injection, and then maybe induced by a variety of means (e.g., change in temperature, pH,exposure to light) to form degradable hydrogel networks in situ or invivo.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures.

In many of the abovementioned embodiments, the framework may be moldedinto a useful shape. Furthermore, it will be appreciated that PPDCs maybe cultured on pre-formed, non-degradable surgical or implantabledevices, e.g., in a manner corresponding to that used for preparingfibroblast-containing GDC endovascular coils, for instance (Marx, W. F.et al., 2001, Am. J. Neuroradiol. 22: 323-333).

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

Frameworks containing living cells are prepared according to methodsknown in the art. For example, cells can be grown freely in a culturevessel to sub-confluency or confluency, lifted from the culture andinoculated onto the framework. Growth factors may be added to theculture medium prior to, during, or subsequent to inoculation of thecells to trigger differentiation and tissue formation, if desired.Alternatively, the frameworks themselves may be modified so that thegrowth of cells thereon is enhanced, or so that the risk of rejection ofthe implant is reduced. Thus, one or more biologically active compounds,including, but not limited to, anti-inflammatory agents,immunosuppressants or growth factors, may be added to the framework forlocal release.

Methods of Use

Postpartum cells, preferably PPDCs, or cell populations, components ofor products produced by such cells, may be used in a variety of ways tosupport and facilitate repair and regeneration of ocular cells andtissues. Such utilities encompass in vitro, ex vivo and in vivo methods.The methods set forth below are directed to PPDCs, but other postpartumcells may also be suitable for use in those methods.

In Vitro and Ex Vivo Methods

In one embodiment, PPDCs may be used in vitro to screen a wide varietyof compounds for effectiveness and cytotoxicity of pharmaceuticalagents, growth factors, regulatory factors, and the like. For example,such screening may be performed on substantially homogeneous populationsof PPDCs to assess the efficacy or toxicity of candidate compounds to beformulated with, or co-administered with, the PPDCs, for treatment of aan ocular condition. Alternatively, such screening may be performed onPPDCs that have been stimulated to differentiate into a cell type foundin the eye, or progenitor thereof, for the purpose of evaluating theefficacy of new pharmaceutical drug candidates. In this embodiment, thePPDCs are maintained in vitro and exposed to the compound to be tested.The activity of a potentially cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth or regulatory factorsmay be assessed by analyzing the number or robustness of the culturedcells, as compared with cells not exposed to the factors. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens.

In a further embodiment, as discussed above, PPDCs can be cultured invitro to produce biological products that are either naturally producedby the cells, or produced by the cells when induced to differentiateinto other lineages, or produced by the cells via genetic modification.For instance, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1,RANTES, 1309, TARC, MDC, and IL-8 were found to be secreted fromumbilicus-derived cells grown in Growth Medium. TIMP1, TPO, KGF, HGF,HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were found tobe secreted from placenta-derived PPDCs cultured in Growth Medium (seeExamples). Some of these trophic factors, such as BDNF and IL-6, haveimportant roles in neural regeneration. Other trophic factors, as yetundetected or unexamined, of use in repair and regeneration of oculartissues, are likely to be produced by PPDCs and possibly secreted intothe medium.

In this regard, another embodiment of the invention features use ofPPDCs for production of conditioned medium, either from undifferentiatedPPDCs or from PPDCs incubated under conditions that stimulatedifferentiation. Such conditioned media are contemplated for use in invitro or ex vivo culture of epithelial or neural precursor cells, forexample, or in vivo to support transplanted cells comprising homogeneouspopulations of PPDCs or heterogeneous populations comprising PPDCs andother progenitors, for example.

Yet another embodiment comprises the use of PPCD cell lysates, solublecell fractions or components thereof, or ECM or components thereof, fora variety of purposes. As mentioned above, some of these components maybe used in pharmaceutical compositions. In other embodiments, a celllysate or ECM is used to coat or otherwise treat substances or devicesto be used surgically, or for implantation, or for ex vivo purposes, topromote healing or survival of cells or tissues contacted in the courseof such treatments.

As described in Examples 13 and 15, PPDCs have demonstrated the abilityto support survival, growth and differentiation of adult neuralprogenitor cells when grown in co-culture with those cells. Likewise,the experimental results set forth in Example 18 indicates that PPDCsmay function to support cells of the retina via trophic mechanisms.Accordingly, in another embodiment, PPDCs are used advantageously inco-cultures in vitro to provide trophic support to other cells, inparticular neural cells and neural and ocular progenitors (e.g., neuralstem cells and retinal or corneal epithelial stem cells). Forco-culture, it may be desirable for the PPDCs and the desired othercells to be co-cultured under conditions in which the two cell types arein contact. This can be achieved, for example, by seeding the cells as aheterogeneous population of cells in culture medium or onto a suitableculture substrate. Alternatively, the PPDCs can first be grown toconfluence, and then will serve as a substrate for the second desiredcell type in culture. In this latter embodiment, the cells may furtherbe physically separated, e.g., by a membrane or similar device, suchthat the other cell type may be removed and used separately, followingthe co-culture period. Use of PPDCs in co-culture to promote expansionand differentiation of neural or ocular cell types may findapplicability in research and in clinical/therapeutic areas. Forinstance, PPDC co-culture may be utilized to facilitate growth anddifferentiation of such cells in culture, for basic research purposes orfor use in drug screening assays, for example. PPDC co-culture may alsobe utilized for ex vivo expansion of neural or ocular progenitors forlater administration for therapeutic purposes. For example, neural orocular progenitor cells may be harvested from an individual, expanded exvivo in co-culture with PPDCs, then returned to that individual(autologous transfer) or another individual (syngeneic or allogeneictransfer). In these embodiments, it will be appreciated that, followingex vivo expansion, the mixed population of cells comprising the PPDCsand progenitors could be administered to a patient in need of treatment.Alternatively, in situations where autologous transfer is appropriate ordesirable, the co-cultured cell populations may be physically separatedin culture, enabling removal of the autologous progenitors foradministration to the patient.

In Vivo Methods

As set forth in Examples 16, 17, and 18, PPDCs have been shown to beeffectively transplanted into the body, and to supply lost neural orretinal function in animal models accepted for their predictability ofefficacy in humans. These results support a preferred embodiment of theinvention, wherein PPDCs are used in cell therapy for treating an oculardegenerative condition. Once transplanted into a target location in theeye, PPDCs may themselves differentiate into one or more phenotypes, orthey may provide trophic support for ocular cells in situ, or they mayexert a beneficial effect in both of those fashions, among others.

PPDCs may be administered alone (e.g., as substantially homogeneouspopulations) or as admixtures with other cells. As described above,PPDCs may be administered as formulated in a pharmaceutical preparationwith a matrix or scaffold, or with conventional pharmaceuticallyacceptable carriers. Where PPDCs are administered with other cells, theymay be administered simultaneously or sequentially with the other cells(either before or after the other cells). Cells that may be administeredin conjunction with PPDCs include, but are not limited to, neurons,astrocytes, oligodendrocytes, neural progenitor cells, neural stemcells, ocular progenitor cells, retinal or corneal epithelial stem cellsand/or other multipotent or pluripotent stem cells. The cells ofdifferent types may be admixed with the PPDCs immediately or shortlyprior to administration, or they may be co-cultured together for aperiod of time prior to administration.

The PPDCs may be administered with other beneficial drugs, biologicalmolecules, such as growth factors, trophic factors, conditioned medium(from postpartum cells or from progenitor or differentiated cellcultures), or other active agents, such as anti-inflammatory agents,anti-apoptotic agents, antioxidants, growth factors, neurotrophicfactors or neuroregenerative or neuroprotective drugs as known in theart. When PPDCs are administered with other agents, they may beadministered together in a single pharmaceutical composition, or inseparate pharmaceutical compositions, simultaneously or sequentiallywith the other agents (either before or after administration of theother agents).

Examples of other components that may be administered with postpartumcells include, but are not limited to: (1) other neuroprotective orneurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, the cells maybe genetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPDXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (6) local anesthetics,to name a few.

In one embodiment, PPDCs are administered as undifferentiated cells,i.e., as cultured in Growth Medium. Alternatively, PPDCs may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired phenotype.

The cells may be surgically implanted, injected or otherwiseadministered directly or indirectly to the site of ocular damage ordistress. When cells are administered in semi-solid or solid devices,surgical implantation into a precise location in the body is typically asuitable means of administration. Liquid or fluid pharmaceuticalcompositions, however, may be administered to a more general location inthe eye (e.g., topically or intra-ocularly).

Other embodiments encompass methods of treating ocular degenerativeconditions by administering pharmaceutical compositions comprising PPDCcellular components (e.g., cell lysates or components thereof) orproducts (e.g., trophic and other biological factors produced naturallyby PPDCs or through genetic modification, conditioned medium from PPDCculture). Again, these methods may further comprise administering otheractive agents, such as growth factors, neurotrophic factors orneuroregenerative or neuroprotective drugs as known in the art.

Dosage forms and regimes for administering PPDCs or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the ocular degenerativecondition, age, sex, body weight and general medical condition, andother factors known to medical practitioners. Thus, the effective amountof a pharmaceutical composition to be administered to a patient isdetermined by these considerations as known in the art.

It may be desirable or appropriate to pharmacologically immunosuppress apatient prior to initiating cell therapy. This may be accomplishedthrough the use of systemic or local immunosuppressive agents, or it maybe accomplished by delivering the cells in an encapsulated device, asdescribed above. These and other means for reducing or eliminating animmune response to the transplanted cells are known in the art. As analternative, PPDCs may be genetically modified to reduce theirimmunogenicity, as mentioned above.

Survival of transplanted cells in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the tissue andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for neural or ocular cells orproducts thereof, e.g., neurotransmitters. Transplanted cells can alsobe identified by prior incorporation of tracer dyes such as rhodamine-or fluorescein-labeled microspheres, fast blue, ferric microparticles,bisbenzamide or genetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted cells into ocular tissue of asubject can be assessed by examining restoration of the ocular functionthat was damaged or diseased. For example, effectiveness in thetreatment of macular degeneration or other retinopathies may bedetermined by improvement of visual acuity and evaluation forabnormalities and grading of stereoscopic color fundus photographs.(Age-Related Eye Disease Study Research Group, NEI, NIH, AREDS ReportNo. 8, 2001, Arch. Opthalmol. 119: 1417-1436).

Kits and Banks

In another aspect, the invention provides kits that utilize postpartumcells, preferably PPDCs, cell populations, components and productsthereof in various methods for ocular regeneration and repair asdescribed above. Where used for treatment of ocular degenerativeconditions, or other scheduled treatment, the kits may include one ormore cell populations, including at least postpartum cells and apharmaceutically acceptable carrier (liquid, semi-solid or solid). Thekits also optionally may include a means of administering the cells, forexample by injection. The kits further may include instructions for useof the cells. Kits prepared for field hospital use, such as for militaryuse may include full-procedure supplies including tissue scaffolds,surgical sutures, and the like, where the cells are to be used inconjunction with repair of acute injuries. Kits for assays and in vitromethods as described herein may contain, for example, one or more of (1)PPDCs or components or products of PPDCs, (2) reagents for practicingthe in vitro method, (3) other cells or cell populations, asappropriate, and (4) instructions for conducting the in vitro method.

In yet another aspect, the invention also provides for banking oftissues, cells, cellular components and cell populations of theinvention. As discussed above, the cells are readily cryopreserved. Theinvention therefore provides methods of cryopreserving the cells in abank, wherein the cells are stored frozen and associated with a completecharacterization of the cells based on immunological, biochemical andgenetic properties of the cells. The frozen cells can be thawed andexpanded or used directly for autologous, syngeneic, or allogeneictherapy, depending on the requirements of the procedure and the needs ofthe patient. Preferably, the information on each cryopreserved sample isstored in a computer, which is searchable based on the requirements ofthe surgeon, procedure and patient with suitable matches being madebased on the characterization of the cells or populations. Preferably,the cells of the invention are grown and expanded to the desiredquantity of cells and therapeutic cell compositions are prepared eitherseparately or as co-cultures, in the presence or absence of a matrix orsupport. While for some applications it may be preferable to use cellsfreshly prepared, the remainder can be cryopreserved and banked byfreezing the cells and entering the information in the computer toassociate the computer entry with the samples. Even where it is notnecessary to match a source or donor with a recipient of such cells, forimmunological purposes, the bank system makes it easy to match, forexample, desirable biochemical or genetic properties of the banked cellsto the therapeutic needs. Upon matching of the desired properties with abanked sample, the sample is retrieved and prepared for therapeutic use.Cell lysates, ECM or cellular components prepared as described hereinmay also be cryopreserved or otherwise preserved (e.g., bylyophilization) and banked in accordance with the present invention.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

As used in the following examples and elsewhere in the specification,the term Growth Medium generally refers to a medium sufficient for theculturing of PPDCs. In particular, one presently preferred medium forthe culturing of the cells of the invention in comprises Dulbecco'sModified Essential Media (also abbreviated DMEM herein). Particularlypreferred is DMEM-low glucose (also DMEM-LG herein) (Invitrogen,Carlsbad, Calif.). The DMEM-low glucose is preferably supplemented with15% (v/v) fetal bovine serum (e.g. defined fetal bovine serum, Hyclone,Logan Utah), antibiotics/antimycotics ((preferably 50-100Units/milliliter penicillin, 50-100 microgram/milliliter streptomycin,and 0-0.25 microgram/milliliter amphotericin B; Invitrogen, Carlsbad,Calif.)), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). Asused in the Examples below, Growth Medium refers to DMEM-low glucosewith 15% fetal bovine serum and antibiotics/antimycotics (whenpenicillin/streptomycin are included, it is preferably at 50 Um' and 50microgram/ml respectively; when penicillin/streptomycin/amphotericin Bare use, it is preferably at 100 U/ml, 100 microgram/ml and 0.25microgram/ml, respectively). In some cases different growth media areused, or different supplementations are provided, and these are normallyindicated in the text as supplementations to Growth Medium.

The following abbreviations may appear in the examples and elsewhere inthe specification and claims: ANG2 (or Ang2) for angiopoietin 2; APC forantigen-presenting cells; BDNF for brain-derived neurotrophic factor;bFGF for basic fibroblast growth factor; bid (BID) for “bis in die”(twice per day); CK18 for cytokeratin 18; CNS for central nervoussystem; CXC ligand 3 for chemokine receptor ligand 3; DMEM forDulbecco's Minimal Essential Medium; DMEM: lg (or DMEM:Lg, DMEM:LG) forDMEM with low glucose; EDTA for ethylene diamine tetraacetic acid; EGF(or E) for epidermal growth factor; FACS for fluorescent activated cellsorting; FBS for fetal bovine serum; FGF (or F) for fibroblast growthfactor; GCP-2 for granulocyte chemotactic protein-2; GF AP for glialfibrillary acidic protein; HB-EGF for heparin-binding epidermal growthfactor; HCAEC for Human coronary artery endothelial cells; HGF forhepatocyte growth factor; hMSC for Human mesenchymal stem cells;HNF-lalpha for hepatocyte-specific transcription factor; HVVEC for Humanumbilical vein endothelial cells; I309 for a chemokine and the ligandfor the CCR8 receptor;

IGF-1 for insulin-like growth factor 1; 1L-6 for interleukin-6; 1L-8 forinterleukin 8; K19 for keratin 19; K8 for keratin 8; KGF forkeratinocyte growth factor; LIF for leukemia inhibitory factor; MBP formyelin basic protein; MCP-1 for monocyte chemotactic protein 1; MDC formacrophage-derived chemokine; MIPlalpha for macrophage inflammatoryprotein 1 alpha; MIPlbeta for macrophage inflammatory protein 1 beta;MMP for matrix metalloprotease (MMP); MSC for mesenchymal stem cells;NHDF for Normal Human Dermal Fibroblasts; NPE for Neural ProgenitorExpansion media; 04 for oligodendrocyte or glial differentiation marker04; PBMC for Peripheral blood mononuclear cell; PBS for phosphatebuffered saline; PDGFbb for platelet derived growth factor; PO for “peros” (by mouth); PNS for peripheral nervous system; Rantes (or RANTES)for regulated on activation, normal T cell expressed and secreted;rhGDF-5 for recombinant human growth and differentiation factor 5; SCfor subcutaneously; SDF-lalpha for stromal-derived factor 1 alpha; SHHfor sonic hedgehog; SOP for standard operating procedure; TARC forthymus and activation-regulated chemokine; TCP for Tissue cultureplastic; TCPS for tissue culture polystyrene; TGFbeta2 for transforminggrowth factor beta2; TGF beta-3 for transforming growth factor beta-3;TIMP1 for tissue inhibitor of matrix metalloproteinase 1; TPO forthrombopoietin; TUJ1 for BIII Tubulin; VEGF for vascular endothelialgrowth factor; vWF for von Willebrand factor; and alphaFP foralpha-fetoprotein.

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLE 1 Derivation of Cells from Postpartum Tissue

This example describes the preparation of postpartum-derived cells fromplacental and umbilical cord tissues. Postpartum umbilical cords andplacentae were obtained upon birth of either a full term or pre-termpregnancy. Cells were harvested from five separate donors of umbilicusand placental tissue. Different methods of cell isolation were testedfor their ability to yield cells with: 1) the potential to differentiateinto cells with different phenotypes, a characteristic common to stemcells, or 2) the potential to provide trophic factors useful for othercells and tissues.

Methods & Materials

Umbilical cell isolation: Umbilical cords were obtained from NationalDisease Research Interchange (NDR1, Philadelphia, Pa.). The tissues wereobtained following normal deliveries. The cell isolation protocol wasperformed aseptically in a laminar flow hood. To remove blood anddebris, the cord was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (100 units/milliliter penicillin, 100 micrograms/milliliterstreptomycin, 0.25 micrograms/milliliter amphotericin B). The tissueswere then mechanically dissociated in 150 cm² tissue culture plates inthe presence of 50 milliliters of medium (DMEM-Low glucose or DMEM-Highglucose; Invitrogen), until the tissue was minced into a fine pulp. Thechopped tissues were transferred to 50 milliliter conical tubes(approximately 5 grams of tissue per tube). The tissue was then digestedin either DMEM-Low glucose medium or DMEM-High glucose medium, eachcontaining antimycotic and antibiotic as described above. In someexperiments, an enzyme mixture of collagenase and dispase was used(“C:D;” collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; anddispase (Invitrogen), 50 Units/milliliter in DMEM:-Low glucose medium).In other experiments a mixture of collagenase, dispase and hyaluronidase(“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter, inDMEM:-Low glucose). The conical tubes containing the tissue, medium anddigestion enzymes were incubated at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,and the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth Medium (DMEM: Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 1milliliter per 100 milliliters of antibiotic/antimycotic as describedabove. The cell suspension was filtered through a 70-micrometer nyloncell strainer (BD Biosciences). An additional 5 milliliters rinsecomprising Growth Medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences) and chased with a rinse of an additional 5 millilitersof Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh Growth Medium. Thenumber of viable cells was determined using Trypan Blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cords were seeded at 5,000 cells/cm²onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning, N.Y.) inGrowth Medium with antibiotics/antimycotics as described above. After 2days (in various experiments, cells were incubated from 2-4 days), spentmedium was aspirated from the flasks. Cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with Growth Medium and allowed to grow to confluence (about10 days from passage 0) to passage 1. On subsequent passages (frompassage 1 to 2 and so on), cells reached sub-confluence (75-85 percentconfluence) in 4-5 days. For these subsequent passages, cells wereseeded at 5000 cells/cm². Cells were grown in a humidified incubatorwith 5 percent carbon dioxide and atmospheric oxygen, at 37° C.

Placental Cell Isolation: Placental tissue was obtained from NDR1(Philadelphia, Pa.). The tissues were from a pregnancy and were obtainedat the time of a normal surgical delivery. Placental cells were isolatedas described for umbilical cell isolation.

The following example applies to the isolation of separate populationsof maternal-derived and neonatal-derived cells from placental tissue.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (as described above) to remove blood and debris. Theplacental tissue was then dissected into three sections: top-line(neonatal side or aspect), mid-line (mixed cell isolation neonatal andmaternal) and bottom line (maternal side or aspect).

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. Eachsection was then mechanically dissociated in 150 cm² tissue cultureplates in the presence of 50 milliliters of DMEM/Low glucose, to a finepulp. The pulp was transferred to 50 milliliter conical tubes. Each tubecontained approximately 5 grams of tissue. The tissue was digested ineither DMEM-Low glucose or DMEM-High glucose medium containingantimycotic and antibiotic (100 U/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B) and digestion enzymes. In some experiments an enzymemixture of collagenase and dispase (“C:D”) was used containingcollagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase(Invitrogen) at 50 Units/milliliter in DMEM-Low glucose medium. In otherexperiments a mixture of collagenase, dispase and hyaluronidase (C:D:H)was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter inDMEM-Low glucose). The conical tubes containing the tissue, medium, anddigestion enzymes were incubated for 2 h at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the resultant supernatant was aspirated off. The pellet was resuspendedin 20 milliliters of Growth Medium withpenicillin/streptomycin/amphotericin B. The cell suspension was filteredthrough a 70 micrometer nylon cell strainer (BD Biosciences), chased bya rinse with an additional 5 milliliters of Growth Medium. The totalcell suspension was passed through a 40 micrometer nylon cell strainer(BD Biosciences) followed with an additional 5 milliliters of GrowthMedium as a rinse.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cell pellet was resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more. After the finalcentrifugation, supernatant was aspirated and the cell pellet wasresuspended in 5 milliliters of fresh Growth Medium. A cell count wasdetermined using the Trypan Blue Exclusion test. Cells were thencultured at standard conditions.

LIBERASE Cell Isolation: Cells were isolated from umbilicus tissues inDMEM-Low glucose medium with LIBERASE (Boehringer Mannheim Corp.,Indianapolis, Ind.) (2.5 milligrams per milliliter, Blendzyme^(®) 3;Roche Applied Sciences, Indianapolis, Ind.) and hyaluronidase (5Units/milliliter, Sigma). Digestion of the tissue and isolation of thecells was as described for other protease digestions above, using theLIBERASE/hyaluronidase mixture in place of the C:D or C:D:H enzymemixture. Tissue digestion with LIBERASE resulted in the isolation ofcell populations from postpartum tissues that expanded readily.

Cell isolation using other enzyme combinations: Procedures were comparedfor isolating cells from the umbilical cord using differing enzymecombinations. Enzymes compared for digestion included: i) collagenase;ii) dispase; iii) hyaluronidase; iv) collagenase: dispase mixture (C;D);v) collagenase: hyaluronidase mixture (C:H); yl) dispase: hyaluronidasemixture (D:H); and vii) collagenase: dispase: hyaluronidase mixture(C:D:H). Differences in cell isolation utilizing these different enzymedigestion conditions were observed (Table 1-1).

Isolation of cells from residual blood in the cords: Other attempts weremade to isolate pools of cells from umbilical cord by differentapproaches. In one instance umbilical cord was sliced and washed withGrowth Medium to dislodge the blood clots and gelatinous material. Themixture of blood, gelatinous material and Growth Medium was collectedand centrifuged at 150×g. The pellet was resuspended and seeded ontogelatin-coated flasks in Growth Medium. From these experiments a cellpopulation was isolated that readily expanded.

Isolation of cells from cord blood: Cells have also been isolated tromcord blood samples attained from NDR1. The isolation protocol used herewas that of International Patent Application PCT/US2002/029971 by Ho etal (Ho, T. W. et al., W02003025149A2). Samples (50 milliliter and 10.5milliliters, respectively) of umbilical cord blood (NDR1, PhiladelphiaPa.) were mixed with lysis buffer (filter-sterilized 155 mM ammoniumchloride, 10 millimolar potassium bicarbonate, 0.1 millimolar EDT Abuffered to pH 7 .2 (all components from Sigma, St. Louis, Mo.)). Cellswere lysed at a ratio of 1 :20 cord blood to lysis buffer. The resultingcell suspension was vortexed for 5 seconds, and incubated for 2 minutesat ambient temperature. The lysate was centrifuged (10 minutes at200^(×)g). The cell pellet was resuspended in complete minimal essentialmedium (Gibco, Carlsbad Calif.) containing 10 percent fetal bovine serum(Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech Herndon, Va.),100 Units penicillin per 100 milliliters and 100 micrograms streptomycinper 100 milliliters (Gibco, Carlsbad, Calif.). The resuspended cellswere centrifuged (10 minutes at 200^(×)g), the supernatant wasaspirated, and the cell pellet was washed in complete medium. Cells wereseeded directly into either T75 flasks (Corning, N.Y.), T75laminin-coated flasks, or T175 fibronectin-coated flasks (both BectonDickinson, Bedford, Mass.).

Isolation of cells using different enzyme combinations and growthconditions: To determine whether cell populations could be isolatedunder different conditions and expanded under a variety of conditionsimmediately after isolation, cells were digested in Growth Medium withor without 0.001 percent (v/v) 2-mercaptoethanol (Sigma, St. Louis,Mo.), using the enzyme combination of C:D:H, according to the proceduresprovided above. Placental-derived cells so isolated were seeded under avariety of conditions. All cells were grown in the presence ofpenicillin/streptomycin. (Table 1-2).

Isolation of cells using different enzyme combinations and growthconditions: In all conditions cells attached and expanded well betweenpassage 0 and 1 (Table 1-2). Cells in conditions 5-8 and 13-16 weredemonstrated to proliferate well up to 4 passages after seeding at whichpoint they were cryopreserved and banked.

Results

Cell isolation using different enzyme combinations: The combination ofC:D:H, provided the best cell yield following isolation, and generatedcells, which expanded for many more generations in culture than theother conditions (Table 1-1). An expandable cell population was notattained using collagenase or hyaluronidase alone. No attempt was madeto determine if this result is specific to the collagen that was tested.

Isolation of cells using different enzyme combinations and growthconditions: Cells attached and expanded well between passage 0 and 1under all conditions tested for enzyme digestion and growth (Table 1-2).Cells in experimental conditions 5-8 and 13-16 proliferated well up to 4passages after seeding, at which point they were cryopreserved. Allcells were banked for further investigation.

Isolation of cells from residual blood in the cords: Nucleated cellsattached and grew rapidly. These cells were analyzed by flow cytometryand were similar to cells obtained by enzyme digestion.

Isolation of cells from cord blood: The preparations contained red bloodcells and platelets. No nucleated cells attached and divided during thefirst 3 weeks. The medium was changed 3 weeks after seeding and no cellswere observed to attach and grow.

Summary: Populations of cells can be derived from umbilical cord andplacental tissue efficiently using the enzyme combination collagenase (amatrix metalloprotease), dispase (a neutral protease) and hyaluronidase(a mucolytic enzyme that breaks down hyaluronic acid). LIBERASE, whichis a Blendzyme®, may also be used. Specifically, Blendzyme®3 which iscollagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) wasalso used together with hyaluronidase to isolate cells. These cellsexpanded readily over many passages when cultured in Growth Medium ongelatin-coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue thatadhere and grow under the conditions used may be due to cells beingreleased during the dissection process.

EXAMPLE 2 Growth Characteristics of Postpartum-Derived Cells

The cell expansion potential of postpartum-derived cells (PPDCs) wascompared to other populations of isolated stem cells. The process ofcell expansion to senescence is referred to as Hayflick's limit(Hayflick L. 1974a, 1974b). Postpartum-derived cells are highly suitedfor therapeutic use because they can be readily expanded to sufficientcell numbers.

Materials and Methods

Gelatin-coating flasks: Tissue culture plastic flasks were coated byadding 20 milliliters 2% (w/v) porcine gelatin (Type B: 225 Bloom;Sigma, St Louis, Mo.) to a T75 flask (Corning, Corning, N.Y.) for 20minutes at room temperature. After removing the gelatin solution, 10milliliters phosphate-buffered saline (PBS) (Invitrogen, Carlsbad,Calif.) was added and then aspirated.

Comparison of expansion potential of PPDCs with other cell populations:For comparison of growth expansion potential the following cellpopulations were utilized; i) Mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat. No. 6,555,374B1; U.S. Patent Application US20040058412); iii) Normal dermal skinfibroblasts (cc-2509 lot #9F0844; Cambrex, Walkersville, Md.); iv)Umbilicus-derived cells; and v) Placenta-derived cells. Cells wereinitially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with penicillin/streptomycin/amphotericin B. Forsubsequent passages, cell cultures were treated as follows. Aftertrypsinization, viable cells were counted after Trypan Blue stainingCell suspension (50 microliters) was combined with Trypan Blue (50milliliters, Sigma, St. Louis Mo.). Viable cell numbers were estimatedusing a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh Growth Medium.Cells were grown under standard conditions at 37° C. The Growth Mediumwas changed twice per week. When cells reached about 85 percentconfluence they were passaged; this process was repeated until the cellsreached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [ln(cell final/cell initial)/ln 2] anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cell final/cell initial).

Expansion potential of cell banks at low density: The expansionpotential of cells banked at passage 10 was also tested, using adifferent set of conditions. Normal dermal skin fibroblasts (cc-2509 lot#9F0844; Cambrex, Walkersville, Md.), umbilicus-derived cells, andplacenta-derived cells were tested. These cell populations had beenbanked at passage 10 previously, having been cultured at 5,000 cells/cm²and grown to confluence at each passage to that point. The effect ofcell density on the cell populations following cell thaw at passage 10was determined Cells were thawed under standard conditions and countedusing Trypan Blue staining. Thawed cells were then seeded at 1000cells/cm² in DMEM:Low glucose Growth Medium with antibiotic/antimycoticas described above. Cells were grown under standard atmosphericconditions at 37° C. Growth Medium was changed twice a week and cellswere passaged as they reached about 85% confluence. Cells weresubsequently passaged until senescence, i.e., until they could not beexpanded any further. Cells were trypsinized and counted at eachpassage. The cell yield, population doubling (ln(cell final/cellinitial)/ln 2) and doubling time (time in culture (h)/populationdoubling). The total cell yield per passage was determined bymultiplying total yield for the previous passage by the expansion factorfor each passage (i.e., expansion factor=cell final/cell initial).

Expansion of PPDCs at low density from initial cell seeding: Theexpansion potential of freshly isolated PPDCs under low cell seedingconditions was tested. PPDDs were prepared as described herein. Cellswere seeded at 1000 cells/cm² and passaged as described above untilsenescence. Cells were grown under standard atmospheric conditions at37° C. Growth Medium was changed twice per week. Cells were passaged asthey reached about 85% confluence. At each passage, cells weretrypsinized and counted by Trypan Blue staining. The cell yield,population doubling (in (cell final/cell initial)/ln 2) and doublingtime (time in culture (h)/population doubling) were calculated for eachpassage. The total cell yield per passage was determined by multiplyingthe total yield for the previous passage by the expansion factor foreach passage (i.e. expansion factor=cell final/cell initial). Cells weregrown on gelatin and non-gelatin coated flasks.

Expansion of clonal neonatal placenta-derived cells: Cloning was used inorder to expand a population of neonatal cells from placental tissue.Following isolation of three differential cell populations from theplacenta (as described herein), these cell populations were expandedunder standard growth conditions and then karyotyped to reveal theidentity of the isolated cell populations. Because the cells wereisolated from a mother who delivered a boy, it was straightforward todistinguish between the male and female chromosomes by performingmetaphase spreads. These experiments demonstrated that fetal-aspectcells were karyotype positive for neonatal phenotype, mid-layer cellswere karyotype positive for both neonatal and maternal phenotypes andmaternal-aspect cells were karyotype positive for maternal cells.

Expansion of cells in low oxygen culture conditions: It has beendemonstrated that low oxygen cell culture conditions can improve cellexpansion in certain circumstances (US20040005704). To determine if cellexpansion of PPDCs could be improved by altering cell cultureconditions, cultures of umbilical-derived cells were grown in low oxygenconditions. Cells were seeded at 5000 cells/cm² in Growth Medium ongelatin coated flasks. Cells were initially cultured under standardatmospheric conditions through passage 5, at which point they weretransferred to low oxygen (5% O₂) culture conditions.

Other growth conditions: In other protocols, cells were expanded onnon-coated, collagen-coated, fibronectin-coated, laminin-coated andextracellular matrix protein-coated plates. Cultures have beendemonstrated to expand well on these different matrices.

Results

Comparison of expansion potential of PPDCs with other stem cell andnon-stem cell populations: Both umbilical-derived and placenta-derivedcells expanded for greater than 40 passages generating cell yields ofgreater than 1E¹⁷ cells in 60 days. In contrast, MSCs and fibroblastssenesced after less than 25 days and less than 60 days, respectively.Although adipose-derived cells expanded for almost 60 days theygenerated total cell yields of 4.5 E¹². Thus, when seeded at 5000cells/cm² under the experimental conditions utilized, postpartum-derivedcells expanded much better than the other cell types grown under thesame conditions (Table 2-1).

Expansion potential of cell banks at low density: Umbilicus-derived,placenta-derived and fibroblast cells expanded for greater than 10passages generating cell yields of greater than 1 E¹¹ cells in 60 days(Table 2-2). After 60 days under these conditions the fibroblasts becamesenescent whereas the umbilicus-derived and placenta-derived cellpopulations senesced after 80 days, completing greater than 50 andgreater than 40 population doublings respectively.

Expansion of PPDCs at low density from initial cell seeding. PPDCs wereexpanded at low density (1,000 cells/cm²) on gelatin-coated and uncoatedplates or flasks. Growth potential of these cells under these conditionswas good. The cells expanded readily in a log phase growth. The rate ofcell expansion was similar to that observed when placenta-derived cellswere seeded at 5000 cells/cm² on gelatin-coated flasks in Growth Medium.No differences were observed in cell expansion potential betweenculturing on either uncoated flasks or gelatin-coated flasks. However,cells appeared phenotypically much smaller on gelatin-coated flasks andmore larger cell phenotypes were observed on uncoated flasks.

Expansion of clonal neonatal or maternal placenta-derived cells: Aclonal neonatal or maternal cell population can be expanded fromplacenta-derived cells isolated from the neonatal aspect or the maternalaspect, respectively, of the placenta. Cells are serially diluted andthen seeded onto gelatin-coated plates in Growth medium for expansion at1 cell/well in 96-well gelatin coated plates. From this initial cloning,expansive clones are identified, trypsinized, and reseeded in 12-wellgelatin-coated plates in Growth medium and then subsequently passagedinto T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium.Subcloning is performed to ensure that a clonal population of cells hasbeen identified. For subcloning experiments, cells are trypsinized andreseeded at 0.5 cells/well. The subclones that grow well are expanded ingelatin-coated T25 flasks at 5, 000 cells cm²/flask. Cells are passagedat 5,000 cells cm²/T75 flask. The growth characteristics of a clone maybe plotted to demonstrate cell expansion. Karyotyping analysis canconfirm that the clone is either neonatal or maternal.

Expansion of cells in low oxygen culture conditions: Cells expanded wellunder the reduced oxygen conditions, however, culturing under low oxygenconditions did not appear to have a significant effect on cell expansionof PPDCs under the conditions used.

Summary: Cell expansion conditions comprising growing isolatedpostpartum-derived cells at densities of about 5000 cells/cm², in GrowthMedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1000 cells/cm²).Postpartum-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing postpartum-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,postpartum-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, postpartum-derived cells expandreadily to large numbers.

EXAMPLE 3 Evaluation of Growth Media for Placenta-Derived Cells

Several cell culture media were evaluated for their ability to supportthe growth of placenta-derived cells. The growth of placenta-derivedcells in normal (20%) and low (5%) oxygen was assessed after 3 daysusing the MTS calorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/wellin 96 well plates in Growth Medium with penicillin/streptomycin. After 8hours the medium was changed as described below and cells were incubatedin normal (atmospheric) or low (5%, v/v) oxygen at 37° C., 5% CO₂ for 48hours. MTS was added to the culture medium CellTiter 96® AQueous OneSolution Cell Proliferation Assay, Promega, Madison, Wis.) for 3 hoursand the absorbance measured at 490 nanometers (Molecular Devices,Sunnyvale Calif.).

Results

Standard curves for the MTS assay established a linear correlationbetween an increase in absorbance and an increase in cell number. Theabsorbance values obtained were converted into estimated cell numbersand the change (%) relative to the initial seeding was calculated.

The Effect of Serum: The addition of serum to media at normal oxygenconditions resulted in a reproducible dose-dependent increase inabsorbance and thus the viable cell number. The addition of serum tocomplete MSCGM resulted in a dose-dependent decrease in absorbance. Inthe media without added serum, cells only grew appreciably in cellgro®FREE™, Ham's F10 and DMEM.

The Effect of Oxygen: Reduced oxygen appeared to increase the growthrate of cells in Growth Medium, Ham's F10, and MSCGM. In decreasingorder of growth, the media resulting in the best growth of the cellswere Growth Medium, greater than MSCGM, greater than Iscove's+10% FBS,equal to DMEM-H+10% FBS, equal to Ham's F12+10% FBS, equal to RPMI1640+10% FBS.

Summary: Placenta-derived cells may be grown in a variety of culturemedia in normal or low oxygen. Short-term growth of placenta-derivedcells was determined in twelve basal media with 0,2 and 10% (v/v) serumin 5% or atmospheric oxygen. In general, placenta-derived cells did notgrow as well in serum-free conditions with the exception of Ham's F10and cellgro® FREE™, which are also protein-free. Growth in theseserum-free media was about 25-33% of the maximal growth observed withmedia containing 15% serum.

EXAMPLE 4 Growth of Postpartum-Derived Cells in Medium ContainingD-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). It was not previously known whether postpartum-derived cellscould grow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9) and umbilical-derivedcells (P5) were seeded at 5×10³ cells/cm² in gelatin-coated T75 flasks(Corning, Corning, N.Y.). After 24 hours the medium was removed and thecells were washed with phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) to remove residual medium. The medium was replaced with aModified Growth Medium (DMEM with D-valine (special order Gibco), 15%(v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma), penicillin/streptomycin (Gibco)).

Results

Placenta-derived, umbilical-derived, and fibroblast cells seeded in theD-valine-containing medium did not proliferate, unlike cells seeded inGrowth Medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after 4 weeks. Theseresults indicate that medium containing D-valine is not suitable forselectively growing postpartum-derived cells.

EXAMPLE 5 Cryopreservation Media for Placenta-Derived Cells

Cryopreservation media for the cryopreservation of placenta-derivedcells were evaluated.

Methods & Materials

Placenta-derived cells grown in Growth Medium in a gelatin-coated T75flask were washed with PBS and trypsinized using 1 milliliterTrypsin/EDTA (Gibco). The trypsinization was stopped by adding 10milliliters Growth Medium. The cells were centrifuged at 150×g,supernatant removed, and the cell pellet was resuspended in 1 milliliterGrowth Medium. An aliquot of cell suspension, 60 microliters, wasremoved and added to 60 microliters trypan blue (Sigma). The viable cellnumber was estimated using a hemocytometer. The cell suspension wasdivided into four equal aliquots each containing 88×10⁴ cells each. Thecell suspension was centrifuged and resuspended in 1 milliliter of eachmedia below and transferred into Cryovials (Nalgene).

-   -   a. Growth Medium+10% (v/v) DMSO (Hybrimax, Sigma, St. Louis,        Mo.)    -   b. Cell Freezing medium w/DMSO, w/methyl cellulose, serum-free        (C6295, Sigma, St. Louis, Mo.)    -   c. Cell Freezing medium serum-free (C2639, Sigma, St. Louis,        Mo.)    -   d. Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis,        Mo.)

The cells were cooled at approximately−1° C/min overnight in a−80° C.freezer using a “Mr Frosty®” freezing container according to themanufacturer's instructions (Nalgene, Rochester, N.Y.). Vials of cellswere transferred into liquid nitrogen for 2 days before thawing rapidlyin a 37° C. water bath. The cells were added to 10 milliliters GrowthMedium and centrifuged before the cell number and viability wasestimated. Cells were seeded onto gelatin-coated flasks at 5,000cells/cm² to determine whether the cells would attach and proliferate.

Results

The initial viability of the cells to be cryopreserved was assessed bytrypan blue staining to be 100%. The initial viability of the cells tobe cryopreserved was assessed by trypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability forC6295 due to cells lysis. The viable cells cryopreserved in all foursolutions attached, divided, and produced a confluent monolayer within 3days. There was no discernable difference in estimated growth rate.

Summary: The cryopreservation of cells is one procedure available forpreparation of a cell bank or a cell product. Four cryopreservationmixtures were compared for their ability to protect humanplacenta-derived cells from freezing damage. Dulbecco's modified Eagle'smedium (DMEM) and 10% (v/v) dimethylsulfoxide (DMSO) is the preferredmedium of those compared for cryopreservation of placenta-derived cells.

EXAMPLE 6 Karyotype Analysis of Postpartum-Derived Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Cells used in cell therapy should have anormal chromosome number (46) and structure. To identify placenta- andumbilicus-derived cell lines that are homogeneous and free from cells ofnon-postpartum tissue origin, karyotypes of cell samples were analyzed.

Methods & Materials

PPDCs from postpartum tissue of a male neonate were cultured in GrowthMedium containing penicillin/streptomycin. Postpartum tissue from a maleneonate (X,Y) was selected to allow distinction between neonatal-derivedcells and maternal derived cells (X,X). Cells were seeded at 5,000 cellsper square centimeter in Growth Medium in a T25 flask (Corning, Corning,N.Y.) and expanded to 80% confluence. A T25 flask containing cells wasfilled to the neck with Growth Medium. Samples were delivered to aclinical cytogenetics laboratory by courier (estimated lab to labtransport time is one hour). Cells were analyzed during metaphase whenthe chromosomes are best visualized. Of twenty cells in metaphasecounted, five were analyzed for normal homogeneous karyotype number(two). A cell sample was characterized as homogeneous if two karyotypeswere observed. A cell sample was characterized as heterogeneous if morethan two karyotypes were observed. Additional metaphase cells werecounted and analyzed when a heterogeneous karyotype number (four) wasidentified.

Results

All cell samples sent for chromosome analysis were interpreted asexhibiting a normal appearance. Three of the sixteen cell lines analyzedexhibited a heterogeneous phenotype (XX and XY) indicating the presenceof cells derived from both neonatal and maternal origins (Table 6-1).Cells derived from tissue Placenta-N were isolated from the neonatalaspect of placenta. At passage zero, this cell line appeared homogeneousXY. However, at passage nine, the cell line was heterogeneous (XX/XY),indicating a previously undetected presence of cells of maternal origin.

Summary: Chromosome analysis identified placenta- and umbilicus-derivedcells whose karyotypes appeared normal as interpreted by a clinicalcytogenetic laboratory. Karyotype analysis also identified cell linesfree from maternal cells, as determined by homogeneous karyotype.

EXAMPLE 7 Evaluation of Human Postpartum-Derived Cell Surface Markers byFlow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum-derivedcell (PPDC) lines isolated from the placenta and umbilicus werecharacterized (by flow cytometry), providing a profile for theidentification of these cell lines.

Methods & Materials

Media and culture vessels: Cells were cultured in Growth Medium (GibcoCarlsbad, Calif.) with penicillin/streptomycin. Cells were cultured inplasma-treated T75, T150, and T225 tissue culture flasks (Corning,Corning, N.Y.) until confluent. The growth surfaces of the flasks werecoated with gelatin by incubating 2% (w/v) gelatin (Sigma, St. Louis,Mo.) for 20 minutes at room temperature.

Antibody Staining and flow cytometry analysis: Adherent cells in flaskswere washed in PBS and detached with Trypsin/EDTA. Cells were harvested,centrifuged, and resuspended in 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. In accordance to themanufacture's specifications, antibody to the cell surface marker ofinterest (see below) was added to one hundred microliters of cellsuspension and the mixture was incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were resuspended in 500 microliter PBSand analyzed by flow cytometry. Flow cytometry analysis was performedwith a FACScalibur™instrument (Becton Dickinson, San Jose, Calif.).Table 7 lists the antibodies to cell surface markers that were used.

Placenta and umbilicus comparison: Placenta-derived cells were comparedto umbilicus-derive cells at passage 8.

Passage to passage comparison: Placenta- and umbilicus-derived cellswere analyzed at passages 8, 15, and 20.

Donor to donor comparison: To compare differences among donors,placenta-derived cells from different donors were compared to eachother, and umbilicus-derived cells from different donors were comparedto each other.

Surface coating comparison: Placenta-derived cells cultured ongelatin-coated flasks was compared to placenta-derived cells cultured onuncoated flasks. Umbilicus-derived cells cultured on gelatin-coatedflasks was compared to umbilicus-derived cells cultured on uncoatedflasks.

Digestion enzyme comparison: Four treatments used for isolation andpreparation of cells were compared. Cells isolated from placenta bytreatment with 1) collagenase; 2) collagenase/dispase; 3)collagenase/hyaluronidase; and 4) collagenase/hyaluronidase/dispase werecompared.

Placental layer comparison: Cells derived from the maternal aspect ofplacental tissue were compared to cells derived from the villous regionof placental tissue and cells derived from the neonatal fetal aspect ofplacenta.

Results

Placenta vs. umbilicus comparison: Placenta- and umbilicus-derived cellsanalyzed by flow cytometry showed positive expression of CD10, CD13,CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated by theincreased values of fluorescence relative to the IgG control. Thesecells were negative for detectable expression of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence valuescomparable to the IgG control. Variations in fluorescence values ofpositive curves were accounted for. The mean (i.e. CD13) and range (i.e.CD90) of the positive curves showed some variation, but the curvesappeared normal, confirming a homogenous population. Both curvesindividually exhibited values greater than the IgG control.

Passage to passage comparison—placenta-derived cells: Placenta-derivedcells at passages 8, 15, and 20 analyzed by flow cytometry all werepositive for expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alphaand HLA-A, B, C, as reflected in the increased value of fluorescencerelative to the IgG control. The cells were negative for expression ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ having fluorescencevalues consistent with the IgG control.

Passage to passage comparison—umbilicus-derived cells: Umbilicus-derivedcells at passage 8, 15, and 20 analyzed by flow cytometry all expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated byincreased fluorescence relative to the IgG control. These cells werenegative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ,indicated by fluorescence values consistent with the IgG control.

Donor to donor comparison—placenta-derived cells: Placenta-derived cellsisolated from separate donors analyzed by flow cytometry each expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, withincreased values of fluorescence relative to the IgG control. The cellswere negative for expression of CD31, CD34, CD45, CD117, CD141, andHLA-DR, DP, DQ as indicated by fluorescence value consistent with theIgG control.

Donor to donor comparison—umbilicus derived cells: Umbilicus-derivedcells isolated from separate donors analyzed by flow cytometry eachshowed positive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alphaand HLA-A, B, C, reflected in the increased values of fluorescencerelative to the IgG control. These cells were negative for expression ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ with fluorescencevalues consistent with the IgG control.

The effect of surface coating with gelatin on placenta-derived cells:Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry all expressed of CD10, CD13, CD44,CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ indicated by fluorescence values consistent with the IgG control.

The effect of surface coating with gelatin on umbilicus-derived cells:Umbilicus-derived cells expanded on gelatin and uncoated flasks analyzedby flow cytometry all were positive for expression of CD10, CD13, CD44,CD73, CD90, PDGFr-alpha and HLA-A, B, C, with increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, withfluorescence values consistent with the IgG control.

Effect of enzyme digestion procedure used for preparation of the cellson the cell surface marker profile: Placenta-derived cells isolatedusing various digestion enzymes analyzed by flow cytometry all expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as indicatedby the increased values of fluorescence relative to the IgG control.These cells were negative for expression of CD31, CD34, CD45, CD117,CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistentwith the IgG control.

Placental layer comparison: Cells isolated from the maternal, villous,and neonatal layers of the placenta, respectively, analyzed by flowcytometry showed positive expression of CD10, CD13, CD44, CD73, CD 90,PDGFr-alpha and HLA-A, B, C, as indicated by the increased value offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ asindicated by fluorescence values consistent with the IgG control.

Summary: Analysis of placenta- and umbilicus-derived cells by flowcytometry has established of an identity of these cell lines. Placenta-and umbilicus-derived cells are positive for CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, HLA-A, B, C and negative for CD31, CD34, CD45, CD117,CD141 and HLA-DR, DP, DQ. This identity was consistent betweenvariations in variables including the donor, passage, culture vesselsurface coating, digestion enzymes, and placental layer. Some variationin individual fluorescence value histogram curve means and ranges wasobserved, but all positive curves under all conditions tested werenormal and expressed fluorescence values greater than the IgG control,thus confirming that the cells comprise a homogenous population that haspositive expression of the markers.

EXAMPLE 8 Immunohistochemical Characterization of Postpartum TissuePhenotypes

The phenotypes of cells found within human postpartum tissues, namelyumbilical cord and placenta, was analyzed by immunohistochemistry.

Methods & Materials

Tissue Preparation: Human umbilical cord and placenta tissue washarvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at4° C. Immunohistochemistry was performed using antibodies directedagainst the following epitopes:vimentin (1:500; Sigma, St. Louis, Mo.),desmin (1:150, raised against rabbit; Sigma; or 1:300, raised againstmouse; Chemicon, Temecula, Calif.), alpha-smooth muscle actin (SMA;1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von WillebrandFactor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100;DAKOCytomation, Carpinteria, Calif.). In addition, the following markerswere tested: anti-human GROalpha—PE (1:100; Becton Dickinson, FranklinLakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz,Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; SantaCruz Biotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 μm thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

Immunohistochemistry Immunohistochemistry was performed similar toprevious studies (e.g., Messina, et al., 2003, Exper. Neurol. 184:816-829). Tissue sections were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 1 hour to access intracellular antigens. Ininstances where the epitope of interest would be located on the cellsurface (CD34, ox-LDL R1), Triton was omitted in all steps of theprocedure in order to prevent epitope loss. Furthermore, in instanceswhere the primary antibody was raised against goat (GCP-2, ox-LDL R1,NOGO-A), 3% (v/v) donkey serum was used in place of goat serumthroughout the procedure. Primary antibodies, diluted in blockingsolution, were then applied to the sections for a period of 4 hours atroom temperature. Primary antibody solutions were removed, and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing block along with goat anti-mouseIgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goatanti-rabbit IgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goatIgG—FITC (1:150; Santa Cruz Biotech). Cultures were washed, and 10micromolar DAPI (Molecular Probes) was applied for 10 minutes tovisualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color video camera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results

Umbilical cord characterization: Vimentin, desmin, SMA, CK18, vWF, andCD34 markers were expressed in a subset of the cells found withinumbilical cord. In particular, vWF and CD34 expression were restrictedto blood vessels contained within the cord. CD34+ cells were on theinnermost layer (lumen side). Vimentin expression was found throughoutthe matrix and blood vessels of the cord. SMA was limited to the matrixand outer walls of the artery & vein, but not contained with the vesselsthemselves. CK18 and desmin were observed within the vessels only,desmin being restricted to the middle and outer layers.

Placenta characterization: Vimentin, desmin, SMA, CK18, vWF, and CD34were all observed within the placenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression: None of thesemarkers were observed within umbilical cord or placental tissue.

Summary: Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18,von Willebrand Factor, and CD34 are expressed in cells within humanumbilical cord and placenta.

EXAMPLE 9 Analysis of Postpartum Tissue-Derived Cells UsingOligonucleotide Arrays

Affymetrix GENECHIP arrays were used to compare gene expression profilesof umbilicus- and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Methods & Materials

Isolation and culture of cells: Human umbilical cords and placenta wereobtained from National Disease Research Interchange (NDR1, Philadelphia,Pa.) from normal full term deliveries with patient consent. The tissueswere received and cells were isolated as described in Example 1. Cellswere cultured in Growth Medium (using DMEM-LG) on gelatin-coated tissueculture plastic flasks. The cultures were incubated at 37° C. with 5%CO₂.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Bothlines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.)with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin(Invitrogen). The cells were grown on standard tissue-treated plastic.

Human mesenchymal stem cells (hMSC) were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human iliac crest bone marrow was received from the NDRI with patientconsent. The marrow was processed according to the method outlined byHo, et al. (W003/025149). The marrow was mixed with lysis buffer (155 mMNH 4C1, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bonemarrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500^(×)g. The supernatant was discarded and the cell pelletwas resuspended in Minimal Essential Medium-alpha (Invitrogen)supplemented with 10% (v/v) fetal bovine serum and 4 mM glutamine. Thecells were centrifuged again and the cell pellet was resuspended infresh medium. The viable mononuclear cells were counted usingtrypan-blue exclusion (Sigma, St. Louis, Mo.). The mononuclear cellswere seeded in tissue-cultured plastic flasks at 5×10⁴ cells/cm². Thecells were incubated at 37° C. with 5% CO₂ at either standardatmospheric O₂ or at 5% O₂. Cells were cultured for 5 days without amedia change. Media and non-adherent cells were removed after 5 days ofculture. The adherent cells were maintained in culture.

Isolation of mRNA and GENECHIP Analysis: Actively growing cultures ofcells were removed from the flasks with a cell scraper in cold PBS. Thecells were centrifuged for 5 minutes at 300³³ g. The supernatant wasremoved and the cells were resuspended in fresh PBS and centrifugedagain. The supernatant was removed and the cell pellet was immediatelyfrozen and stored at−80° C. Cellular mRNA was extracted and transcribedinto cDNA, which was then transcribed into cRNA and biotin-labeled. Thebiotin-labeled cRNA was hybridized with HG-U133A GENECHIPoligonucleotide array (Affymetrix, Santa Clara Calif.). Thehybridization and data collection was performed according to themanufacturer's specifications. Analyses were performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Stanford University; Tusher, V. G. et al., 2001, Proc. Natl.Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed. The cells alongwith passage information, culture substrate, and culture media arelisted in Table 9-1.

The data were evaluated by a Principle Component Analysis, analyzing the290 genes that were differentially expressed in the cells. This analysisallows for a relative comparison for the similarities between thepopulations. Table 9-2 shows the Euclidean distances that werecalculated for the comparison of the cell pairs. The Euclidean distanceswere based on the comparison of the cells based on the 290 genes thatwere differentially expressed among the cell types. The Euclideandistance is inversely proportional to similarity between the expressionof the 290 genes (i.e., the greater the distance, the less similarityexists).

Tables 9-3, 9-4, and 9-5 show the expression of genes increased inplacenta-derived cells (Table 9-3), increased in umbilicus-derived cells(Table 9-4), and reduced in umbilicus- and placenta-derived cells (Table9-5). The column entitled “Probe Set ID” refers to the manufacturer'sidentification code for the sets of several oligonucleotide probeslocated on a particular site on the chip, which hybridize to the namedgene (column “Gene Name”), comprising a sequence that can be foundwithin the NCBI (GenBank) database at the specified accession number(column “NCBI Accession Number”).

Tables 9-6, 9-7, and 9-8 show the expression of genes increased in humanfibroblasts (Table 9-6), ICBM cells (Table 9-7), and MSCs (Table 9-8).

Summary: The present examination was performed to provide a molecularcharacterization of the postpartum cells derived from umbilical cord andplacenta. This analysis included cells derived from three differentumbilical cords and three different placentas. The examination alsoincluded two different lines of dermal fibroblasts, three lines ofmesenchymal stem cells, and three lines of iliac crest bone marrowcells. The mRNA that was expressed by these cells was analyzed using anoligonucleotide array that contained probes for 22,000 genes. Resultsshowed that 290 genes are differentially expressed in these fivedifferent cell types. These genes include ten genes that arespecifically increased in the placenta-derived cells and seven genesspecifically increased in the umbilical cord-derived cells. Fifty-fourgenes were found to have specifically lower expression levels inplacenta and umbilical cord, as compared with the other cell types. Theexpression of selected genes has been confirmed by PCR (see the examplethat follows). These results demonstrate that the postpartum-derivedcells have a distinct gene expression profile, for example, as comparedto bone marrow-derived cells and fibroblasts.

EXAMPLE 10 Cell Markers in Postpartum-Derived Cells

In the preceding example, similarities and differences in cells derivedfrom the human placenta and the human umbilical cord were assessed bycomparing their gene expression profiles with those of cells derivedfrom other sources (using an oligonucleotide array). Six “signature”genes were identified: oxidized LDL receptor 1, interleukin-8, rennin,reticulon, chemokine receptor ligand 3 (CXC ligand 3), and granulocytechemotactic protein 2 (GCP-2). These “signature” genes were expressed atrelatively high levels in postpartum-derived cells.

The procedures described in this example were conducted to verify themicroarray data and find concordance/discordance between gene andprotein expression, as well as to establish a series of reliable assayfor detection of unique identifiers for placenta- and umbilicus-derivedcells.

Methods & Materials

Cells: Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis),umbilicus-derived cells (four isolates), and Normal Human DermalFibroblasts (NHDF; neonatal and adult) grown in Growth Medium withpenicillin/streptomycin in a gelatin-coated T75 flask. Mesechymal StemCells (MSCS) were grown in Mesenchymal Stem Cell Growth Medium Bulletkit (MSCGM; Cambrex, Walkerville, Md.).

For the IL-8 protocol, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth Medium and then grown for further 8 hours in 10 milliliters ofserum starvation medium [DMEM—low glucose (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco, Carlsbad, Calif.) and 0.1% (w/v) BovineSerum Albumin (BSA; Sigma, St. Louis, Mo.)]. After this treatment RNAwas extracted and the supernatants were centrifuged at 150×g for 5minutes to remove cellular debris. Supernatants were then frozen at −80°C. for ELISA analysis.

Cell culture for ELISA assay: Postpartum cells derived from placenta andumbilicus, as well as human fibroblasts derived from human neonatalforeskin were cultured in Growth Medium in gelatin-coated T75 flasks.Cells were frozen at passage 11 in liquid nitrogen. Cells were thawedand transferred to 15-milliliter centrifuge tubes. After centrifugationat 150×g for 5 minutes, the supernatant was discarded. Cells wereresuspended in 4 milliliters culture medium and counted. Cells weregrown in a 75 cm² flask containing 15 milliliters of Growth Medium at375,000 cells/flask for 24 hours. The medium was changed to a serumstarvation medium for 8 hours. Serum starvation medium was collected atthe end of incubation, centrifuged at 14,000×g for 5 minutes (and storedat −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added each flask. After cellsdetached from the flask, trypsin activity was neutralized with 8milliliters of Growth Medium. Cells were transferred to a 15 milliliterscentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth Medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA assay: The amount of IL-8 secreted by the cells into serumstarvation medium was analyzed using ELISA assays (R&D Systems,Minneapolis, Minn.). All assays were tested according to theinstructions provided by the manufacturer.

Total RNA isolation: RNA was extracted from confluent postpartum-derivedcells and fibroblasts or for IL-8 expression from cells treated asdescribed above. Cells were lysed with 350 microliters buffer RLTcontaining beta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (Rneasy® Mini Kit; Qiagen, Valencia, Calif).RNA was extracted according to the manufacturer's instructions (Rneasy®Mini Kit; Qiagen, Valencia, Calif) and subjected to DNase treatment (2.7U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50 microlitersDEPC-treated water and stored at−80° C.

Reverse transcription: RNA was also extracted from human placenta andumbilicus. Tissue (30 milligram) was suspended in 700 microliters ofbuffer RLT containing 2 -mercaptoethanol. Samples were mechanicallyhomogenized and the RNA extraction proceeded according to manufacturer'sspecification. RNA was extracted with 50 microliters of DEPC-treatedwater and stored at−80° C. . RNA was reversed transcribed using randomhexamers with the TaqMan® reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes, and 95° C. for 10 minutes. Samples were stored at−20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (signature genes—including oxidized LDL receptor, interleukin-8,rennin and reticulon), were further investigated using real-time andconventional PCR.

Real-time PCR: PCR was performed on cDNA samples using Assays-on-Demand®gene expression products: oxidized LDL receptor (Hs00234028); rennin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH (Applied Biosystems, FosterCity, Calif.) were mixed with cDNA and TaqMan Universal PCR master mixaccording to the manufacturer's instructions (Applied Biosystems, FosterCity, Calif.) using a 7000 sequence detection system with ABI Prism 7000SDS software (Applied Biosystems, Foster City, Calif.). Thermal cycleconditions were initially 50° C. for 2 min and 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min. PCRdata was analyzed according to manufacturer's specifications (UserBulletin #2 from Applied Biosystems for ABI Prism 7700 SequenceDetection System).

Conventional PCR: Conventional PCR was performed using an ABI PRISM 7700(Perkin Elmer Applied Biosystems, Boston, Mass., USA) to confirm theresults from real-time PCR. PCR was performed using 2 microliters ofcDNA solution, 1×AmpliTaq Gold universal mix PCR reaction buffer(Applied Biosystems, Foster City, Calif.) and initial denaturation at94° C. for 5 minutes. Amplification was optimized for each primer set.For IL-8, CXC ligand 3, and reticulon (94° C. for 15 seconds, 55° C. for15 seconds and 72° C. for 30 seconds for 30 cycles); for rennin (94° C.for 15 seconds, 53° C. for 15seconds and 72° C. for 30 seconds for 38cycles); for oxidized LDL receptor and GAPDH (94° C. for 15 seconds, 55°C. for 15 seconds and 72° C. for 30 seconds for 33 cycles). Primers usedfor amplification are listed in Table 10. Primer concentration in thefinal PCR reaction was 1 micromolar except for GAPDH, which was 0.5micromolar. GAPDH primers were the same as real-time PCR, except thatthe manufacturer's TaqMan® probe was not added to the final PCRreaction. Samples were run on 2% (w/v) agarose gel and stained withethidium bromide (Sigma, St. Louis, Mo.). Images were captured using a667 Universal Twinpack film (VWR International, South Plainfield, N.J.)using a focal length Polaroid camera (VWR International, SouthPlainfield, N.J.).

Immunofluorescence: PPDCs were fixed with cold 4% (w/v) paraformaldehyde(Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at room temperature. Oneisolate each of umbilicus- and placenta-derived cells at passage 0 (P0)(directly after isolation) and passage 11 (P 11) (two isolates ofplacenta-derived, two isolates of umbilicus-derived cells) andfibroblasts (P11) were used Immunocytochemistry was performed usingantibodies directed against the following epitopes:vimentin (1:500,Sigma, St. Louis, Mo.), desmin (1:150; Sigma—raised against rabbit; or1:300; Chemicon, Temecula, Calif—raised against mouse), alpha-smoothmuscle actin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma),von Willebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 ClassIII; 1:100; DAKOCytomation, Carpinteria, Calif.). In addition, thefollowing markers were tested on passage 11 postpartum cells: anti-humanGRO alpha—PE (1:100; Becton Dickinson, Franklin Lakes, N.J.), anti-humanGCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.), anti-humanoxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa Cruz Biotech), andanti-human NOGA-A (1:100; Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Preparation of cells for FACS analysis: Adherent cells in flasks werewashed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif) anddetached with Trypsin/EDTA (Gibco, Carlsbad, Calif). Cells wereharvested, centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cellconcentration of 1×10 7 per milliliter. One hundred microliter aliquotswere delivered to conical tubes. Cells stained for intracellularantigens were permeabilized with Perm/Wash buffer (BD Pharmingen, SanDiego, Calif). Antibody was added to aliquots as per manufacturesspecifications and the cells were incubated for in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove excess antibody. Cells requiring a secondaryantibody were resuspended in 100 microliters of 3% FBS. Secondaryantibody was added as per manufactures specification and the cells wereincubated in the dark for 30 minutes at 4° C. After incubation, cellswere washed with PBS and centrifuged to remove excess secondaryantibody. Washed cells were resuspended in 0.5 milliliters PBS andanalyzed by flow cytometry. The following antibodies were used: oxidizedLDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.). Flowcytometry analysis was performed with FACScalibur™ (Becton Dickinson SanJose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentae, adult and neonatalfibroblasts and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and rennin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the AACT method and expressed on alogarithmic scale. Levels of reticulon and oxidized LDL receptorexpression were higher in umbilicus-derived cells as compared to othercells. No significant difference in the expression levels of CXC ligand3 and GCP-2 were found between postpartum-derived cells and controls.The results of real-time PCR were confirmed by conventional PCR.Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum-derived cells and controls using conventional PCR CXCligand 3 primers listed above.

The production of the cytokine, IL-8 in postpartum was elevated in bothGrowth Medium-cultured and serum-starved postpartum-derived cells. Allreal-time PCR data was validated with conventional PCR and by sequencingPCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, the highest amounts were detected in media derivedfrom umbilical cells and some isolates of placenta cells (Table 10⁻¹).No IL-8 was detected in medium derived from human dermal fibroblasts.

Placenta-derived cells were also examined for the production of oxidizedLDL receptor, GCP-2 and GROalpha by FACS analysis. Cells tested positivefor GCP-2. Oxidized LDL receptor and GRO were not detected by thismethod.

Placenta-derived cells were also tested for the production of selectedproteins by immunocytochemical analysis Immediately after isolation(passage 0), cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed forthe production of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilicus-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

Summary: Concordance between gene expression levels measured bymicroarray and PCR (both real-time and conventional) has beenestablished for four genes: oxidized LDL receptor 1, rennin, reticulon,and IL-8. The expression of these genes was differentially regulated atthe mRNA level in PPDCs, with IL-8 also differentially regulated at theprotein level. The presence of oxidized LDL receptor was not detected atthe protein level by FACS analysis in cells derived from the placenta.Differential expression of GCP-2 and CXC ligand 3 was not confirmed atthe mRNA level, however GCP-2 was detected at the protein level by FACSanalysis in the placenta-derived cells. Although this result is notreflected by data originally obtained from the microarray experiment,this may be due to a difference in the sensitivity of the methodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin expressionmay be preserved in cells with passaging, in the Growth Medium and underthe conditions utilized in these procedures. Cells derived from thehuman umbilical cord at passage 0 were probed for the expression ofalpha-smooth muscle actin and vimentin, and were positive for both. Thestaining pattern was preserved through passage 11.

EXAMPLE 11 In Vitro Immunological Evaluation of Postpartum-Derived Cells

Postpartum-derived cells (PPDCs) were evaluated in vitro for theirimmunological characteristics in an effort to predict the immunologicalresponse, if any, these cells would elicit upon in vivo transplantation.PPDCs were assayed by flow cytometry for the presence of HLA-DR, HLA-DP,HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4+T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the expressionof HLA-G (Abbas & Lichtman, 2003, supra), CD 178 (Coumans, et al.,(1999) Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas& Lichtman, 2003, supra; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The expression of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to which placenta- andumbilicus-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Methods & Materials

Cell culture: Cells were cultured to confluence in Growth Mediumcontaining penicillin/streptomycin in T75 flasks (Corning, Corning,N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining: Cells were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Mo.). Cells were harvested, centrifuged, and re-suspended in3% (v/v) FBS in PBS at a cell concentration of 1×10 ⁷ per milliliter.Antibody (Table 11-1) was added to one hundred microliters of cellsuspension as per manufacturer's specifications and incubated in thedark for 30 minutes at 4° C. . After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were re-suspendedin five hundred microliters of PBS and analyzed by flow cytometry usinga FACSCalibur® instrument (Becton Dickinson, San Jose, Calif.).

Mixed Lymphocyte Reaction: Cryopreserved vials of passage 10umbilicus-derived cells labeled as cell line A and passage 11placenta-derived cells labeled as cell line B were sent on dry ice toCTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction usingCTBR SOP No. CAC-031. Peripheral blood mononuclear cells (PBMCs) werecollected from multiple male and female volunteer donors. Stimulator(donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines weretreated with mitomycin C. Autologous and mitomycin C-treated stimulatorcells were added to responder (recipient) PBMCs and cultured for 4 days.After incubation, [³H]-thymidine was added to each sample and culturedfor 18 hours. Following harvest of the cells, radiolabeled DNA wasextracted, and [³H]-thymidine incorporation was measured using ascintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the PPDCs was calculated as the meanproliferation of the receiver plus mitomycin C-treated postpartum cellline divided by the baseline proliferation of the receiver.

Results

Mixed lymphocyte reaction—placenta-derived cells: Seven human volunteerblood donors were screened to identify a single allogeneic donor thatwould exhibit a robust proliferation response in a mixed lymphocytereaction with the other six blood donors. This donor was selected as theallogeneic positive control donor. The remaining six blood donors wereselected as recipients. The allogeneic positive control donor andplacenta-derived cell lines were treated with mitomycin C and culturedin a mixed lymphocyte reaction with the six individual allogeneicreceivers. Reactions were performed in triplicate using two cell cultureplates with three receivers per plate (Table 11-2). The averagestimulation index ranged from 1.3 (plate 2) to 3 (plate 1) and theallogeneic donor positive controls ranged from 46.25 (plate 2) to 279(plate 1) (Table 11-3).

Mixed lymphocyte reaction—umbilicus-derived cells: Six human volunteerblood donors were screened to identify a single allogeneic donor thatwill exhibit a robust proliferation response in a mixed lymphocytereaction with the other five blood donors. This donor was selected asthe allogeneic positive control donor. The remaining five blood donorswere selected as recipients. The allogeneic positive control donor andplacenta cell lines were mitomycin C-treated and cultured in a mixedlymphocyte reaction with the five individual allogeneic receivers.Reactions were performed in triplicate using two cell culture plateswith three receivers per plate (Table 11-4). The average stimulationindex ranged from 6.5 (plate 1) to 9 (plate 2) and the allogeneic donorpositive controls ranged from 42.75 (plate 1) to 70 (plate 2) (Table11-5).

Antigen presenting cell markers—placenta-derived cells: Histograms ofplacenta-derived cells analyzed by flow cytometry show negativeexpression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted byfluorescence value consistent with the IgG control, indicating thatplacental cell lines lack the cell surface molecules required todirectly stimulate CD4+T cells.

Immunomodulating markers—placenta-derived cells: Histograms ofplacenta-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control.

Antigen presenting cell markers—umbilicus-derived cells: Histograms ofumbilicus-derived cells analyzed by flow cytometry show negativeexpression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted byfluorescence value consistent with the IgG control, indicating thatumbilical cell lines lack the cell surface molecules required todirectly stimulate CD4+T cells.

Immunomodulating cell markers—umbilicus-derived cells: Histograms ofumbilicus-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control.

Summary: In the mixed lymphocyte reactions conducted withplacenta-derived cell lines, the average stimulation index ranged from1.3 to 3, and that of the allogeneic positive controls ranged from 46.25to 279. In the mixed lymphocyte reactions conducted withumbilicus-derived cell lines the average stimulation index ranged from6.5 to 9, and that of the allogeneic positive controls ranged from 42.75to 70. Placenta- and umbilicus-derived cell lines were negative for theexpression of the stimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80,CD86, and B7-H2, as measured by flow cytometry. Placenta- andumbilicus-derived cell lines were negative for the expression ofimmuno-modulating proteins HLA-G and CD 178 and positive for theexpression of PD-L2, as measured by flow cytometry. Allogeneic donorPBMCs contain antigen-presenting cells expressing HLA-DR, DQ, CD8, CD86,and B7-H2, thereby allowing for the stimulation of naïve CD4+T cells.The absence of antigen-presenting cell surface molecules on placenta-and umbilicus-derived cells required for the direct stimulation of naïveCD4+ T cells and the presence of PD-L2, an immunomodulating protein, mayaccount for the low stimulation index exhibited by these cells in a MLRas compared to allogeneic controls.

EXAMPLE 12 Secretion of Trophic Factors by Postpartum-Derived Cells

The secretion of selected trophic factors from placenta-andumbilicus-derived cells was measured. Factors selected for detectionincluded: (1) those known to have angiogenic activity, such ashepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp.212:215-26), monocyte chemotactic protein 1 (MCP-1) (Salcedo et al.(2000) Blood 96;34-40), interleukin-8 (IL-8) (Li et al. (2003) J.Immunol. 170:3369-76), keratinocyte growth factor (KGF), basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), matrixmetalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2), platelet derivedgrowth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermalgrowth factor (HB-EGF), stromal-derived factor lalpha (SDF-lalpha); (2)those known to have neurotrophic/neuroprotective activity, such asbrain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol.258;319-33), interleukin-6 (IL-6), granulocyte chemotactic protein-2(GCP-2), transforming growth factor beta2 (TGFbeta2) ; and (3) thoseknown to have chemokine activity, such as macrophage inflammatoryprotein 1 alpha (MIP 1 a), macrophage inflammatory protein 1 beta (MIP 1b), monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation,normal T cell expressed and secreted),I309, thymus andactivation-regulated chemokine (TARe), Eotaxin, macrophage-derivedchemokine (MDC), IL-8).

Methods & Materials

Cell culture: PPDCs from placenta and umbilicus as well as humanfibroblasts derived from human neonatal foreskin were cultured in GrowthMedium with penicillin/streptomycin on gelatin-coated T75 flasks. Cellswere cryopreserved at passage 11 and stored in liquid nitrogen. Afterthawing of the cells, Growth Medium was added to the cells followed bytransfer to a 15 milliliter centrifuge tube and centrifugation of thecells at 150×g for 5 minutes. The supernatant was discarded. The cellpellet was resuspended in 4 milliliters Growth Medium, and cells werecounted. Cells were seeded at 375,000 cells/75 cm² flask containing 15milliliters of Growth Medium and cultured for 24 hours. The medium waschanged to a serum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v)bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) for 8hours. Conditioned serum-free medium was collected at the end ofincubation by centrifugation at 14,000×g for 5 minutes and stored at−20° C. To estimate the number of cells in each flask, cells were washedwith PBS and detached using 2 milliliters trypsin/EDTA. Trypsin activitywas inhibited by addition of 8 milliliters Growth Medium. Cells werecentrifuged at 150×g for 5 minutes. Supernatant was removed, and cellswere resuspended in 1 milliliter Growth Medium. Cell number wasestimated using a hemocytometer.

ELISA assay: Cells were grown at 37° C. in 5% carbon dioxide andatmospheric oxygen. Placenta-derived cells (batch 101503) also weregrown in 5% oxygen or beta-mercaptoethanol (BME). The amount of MCP-1,IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta 2 produced by eachcell sample was measured by an ELISA assay (R&D Systems, Minneapolis,Minn.). All assays were performed according to the manufacturer'sinstructions.

SearchLight™ multiplexed ELISA assay: Chemokines (MIP1a, MIP1b, MCP-1,Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors(HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF were measuredusing SearchLight™ Proteome Arrays (Pierce Biotechnology Inc.). TheProteome Arrays are multiplexed sandwich ELISAs for the quantitativemeasurement of two to 16 proteins per well. The arrays are produced byspotting a 2×2, 3×3, or 4×4 pattern of four to 16 different captureantibodies into each well of a 96-well plate. Following a sandwich ELISAprocedure, the entire plate is imaged to capture chemiluminescent signalgenerated at each spot within each well of the plate. The amount ofsignal generated in each spot is proportional to the amount of targetprotein in the original standard or sample.

Results

ELISA assay: MCP-1 and IL-6 were secreted by placenta- andumbilicus-derived cells and dermal fibroblasts (Table 12-1). SDF-1alphawas secreted by placenta-derived cells cultured in 5% O2 and byfibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived cells andby placenta-derived cells cultured in the presence of BME or 5% O2.GCP-2 also was secreted by human fibroblasts. TGF-beta2 was notdetectable by ELISA assay.

SearchLight™ multiplexed ELISA assay: TIMP1, TPO, KGF, HGF, FGF, HBEGF,BDNF, MIP1b, MCP1, RANTES, 1309, TARC, MDC, and IL-8 were secreted fromumbilicus-derived cells (Tables 12-2 and 12-3). TIMP1, TPO, KGF, HGF,HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were secretedfrom placenta-derived cells (Tables 12-2 and 12-3). No Ang2, VEGF, orPDGF-bb were detected.

Summary: Umbilicus- and placenta-derived cells secreted a number oftrophic factors. Some of these trophic factors, such as HGF, bFGF, MCP-1and IL-8, play important roles in angiogenesis. Other trophic factors,such as BDNF and IL-6, have important roles in neural regeneration.

EXAMPLE 13 Short-Term Neural Differentiation of Postpartum-Derived Cells

The ability of placenta- and umbilicus-derived cells (collectivelypostpartum-derived cells or PPDCs) to differentiate into neural lineagecells was examined.

Methods & Materials

Isolation and Expansion of Postpartum Cells: PPDCs from placental andumbilical tissues were isolated and expanded as described in Example 1.

Modified Woodbury-Black Protocol (A): This assay was adapted from anassay originally performed to test the neural induction potential ofbone marrow stromal cells (1). Umbilicus-derived cells (022803) P4 andplacenta-derived cells (042203) P3 were thawed and culture expanded inGrowth Media at 5,000 cells/cm² until sub-confluence (75%) was reached.Cells were then trypsinized and seeded at 6,000 cells per well of aTitretek II glass slide (VWR International, Bristol, Conn.). Ascontrols, mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville,Md.), osteoblasts (P5; CC2538; Cambrex), adipose-derived cells (Artecel,U.S. Pat. No. 6,555,374 B1) (P6; Donor 2) and neonatal human dermalfibroblasts (P6; CC2509; Cambrex) were also seeded under the sameconditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20nanograms/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growthfactor (EGF; 20 nanograms/milliliter; Peprotech) andpenicillin/streptomycin (Invitrogen). After four days, cells were rinsedin phosphate-buffered saline (PBS; Invitrogen) and were subsequentlycultured in DMEM/F12 medium+20% (v/v) FBS+penicillin/streptomycin for 24hours. After 24 hours, cells were rinsed with PBS. Cells were thencultured for 1-6 hours in an induction medium which was comprised ofDMEM/F12 (serum-free) containing 200 mM butylated hydroxyanisole, 10 μMpotassium chloride, 5 milligram/milliliter insulin, 10 μM forskolin, 4μM valproic acid, and 2 μM hydrocortisone (all chemicals from Sigma, St.Louis, Mo.). Cells were then fixed in 100% ice-cold methanol andimmunocytochemistry was performed (see methods below) to assess humannestin protein expression.

Modified Woodbury-Black Protocol (B): PPDCs (umbilicus (022803) P11;placenta (042203) P11) and adult human dermal fibroblasts (1F1853, P11)were thawed and culture expanded in Growth Medium at 5,000 cells/cm²until sub-confluence (75%) was reached. Cells were then trypsinized andseeded at similar density as in (A), but onto (1) 24 well tissueculture-treated plates (TCP, Falcon brand, VWR International), (2) TCPwells+2% (w/v) gelatin adsorbed for 1 hour at room temperature, or (3)TCP wells+20 μg/milliliter adsorbed mouse laminin (adsorbed for aminimum of 2 hours at 37° C.; Invitrogen).

Exactly as in (A), cells were initially expanded and media switched atthe aforementioned timeframes. One set of cultures was fixed, as before,at 5 days and six hours, this time with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature. In thesecond set of cultures, medium was removed and switched to NeuralProgenitor Expansion medium (NPE) consisting of Neurobasal-A medium(Invitrogen) containing B27 (B27 supplement; Invitrogen), L-glutamine (4mM), and penicillin/streptomycin (Invitrogen). NPE medium was furthersupplemented with retinoic acid (RA; 1 μM; Sigma). This medium wasremoved 4 days later and cultures were fixed with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature, and stainedfor nestin, GFAP, and TuJ1 protein expression (see Table 13-1).

Two Stage Differentiation Protocol: PPDCs (umbilicus (042203) P11,placenta (022803) P11), adult human dermal fibroblasts (P11; 1F1853;Cambrex) were thawed and culture expanded in Growth Medium at 5,000cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at 2,000 cells/cm², but onto 24 well platescoated with laminin (BD Biosciences, Franklin Lakes, N.J.) in thepresence of NPE media supplemented with bFGF (20 nanograms/milliliter;Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter;Peprotech) [whole media composition further referred to as NPE+F+E]. Atthe same time, adult rat neural progenitors isolated from hippocampus(P4; (062603) were also plated onto 24 well laminin-coated plates inNPE+F+E media. All cultures were maintained in such conditions for aperiod of 6 days (cells were fed once during that time) at which timemedia was switched to the differentiation conditions listed in Table13-2 for an additional period of 7 days. Cultures were fixed withice-cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at roomtemperature, and stained for human or rat nestin, GFAP, and TuJ1 proteinexpression.

Multiple growth factor protocol: Umbilicus-derived cells (P11; (042203))were thawed and culture expanded in Growth Medium at 5,000 cells/cm²until sub-confluence (75%) was reached. Cells were then trypsinized andseeded at 2,000 cells/cm², onto 24 well laminin-coated plates (BDBiosciences) in the presence of NPE+F (20 nanograms/milliliter)+E (20nanograms/milliliter). In addition, some wells contained NPE+F+E+2% FBSor 10% FBS. After four days of “pre-differentiation” conditions, allmedia were removed and samples were switched to NPE medium supplementedwith sonic hedgehog (SHH; 200 nanograms/milliliter; Sigma, St. Louis,Mo.), FGF8 (100 nanograms/milliliter; Peprotech), BDNF (40nanograms/milliliter; Sigma), GDNF (20 nanograms/milliliter; Sigma), andretinoic acid (1 μM; Sigma). Seven days post medium change, cultureswere fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10minutes at room temperature, and stained for human nestin, GFAP, TuJ1 ,desmin, and alpha-smooth muscle actin expression.

Neural progenitor co-culture protocol: Adult rat hippocampal progenitors(062603) were plated as neurospheres or single cells (10,000 cells/well)onto laminin-coated 24 well dishes (BD Biosciences) in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter).

Separately, umbilicus-derived cells (042203) P11 and placenta-derivedcells (022803) P11 were thawed and culture expanded in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter) at 5,000 cells/cm² fora period of 48 hours. Cells were then trypsinized and seeded at 2,500cells/well onto existing cultures of neural progenitors. At that time,existing medium was exchanged for fresh medium. Four days later,cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for10 minutes at room temperature, and stained for human nuclear protein(hNuc; Chemicon) (Table 13-1 above) to identify PPDCs.

Immunocytochemistry: Immunocytochemistry was performed using theantibodies listed in Table 13-1. Cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibodies solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 micromolar DAPI (MolecularProbes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

Modified Woodbury-Black Protocol (A): Upon incubation in this neuralinduction composition, all cell types transformed into cells withbipolar morphologies and extended processes. Other larger non-bipolarmorphologies were also observed. Furthermore, the induced cellpopulations stained positively for nestin, a marker of multipotentneural stem and progenitor cells.

Modified Woodbury-Black Protocol (B): When repeated on tissue cultureplastic (TCP) dishes, nestin expression was not observed unless lamininwas pre-adsorbed to the culture surface. To further assess whethernestin-expressing cells could then go on to generate mature neurons,PPDCs and fibroblasts were exposed to NPE+RA (1 μM), a media compositionknown to induce the differentiation of neural stem and progenitor cellsinto such cells (2, 3, 4). Cells were stained for TuJ1, a marker forimmature and mature neurons, GFAP, a marker of astrocytes, and nestin.Under no conditions was TuJ1 detected, nor were cells with neuronalmorphology observed, suggesting that neurons were not generated in theshort term. Furthermore, nestin and GFAP were no longer expressed byPPDCs, as determined by immunocytochemistry.

Two-stage differentiation: Umbilicus and placenta PPDC isolates (as wellas human fibroblasts and rodent neural progenitors as negative andpositive control cell types, respectively) were plated on laminin(neural promoting)-coated dishes and exposed to 13 different growthconditions (and two control conditions) known to promote differentiationof neural progenitors into neurons and astrocytes. In addition, twoconditions were added to examine the influence of GDF5, and BMP7 on PPDCdifferentiation. Generally, a two-step differentiation approach wastaken, where the cells were first placed in neural progenitor expansionconditions for a period of 6 days, followed by full differentiationconditions for 7 days. Morphologically, both umbilicus- andplacenta-derived cells exhibited fundamental changes in cell morphologythroughout the time-course of this procedure. However, neuronal orastrocytic-shaped cells were not observed except for in control, neuralprogenitor-plated conditions Immunocytochemistry, negative for humannestin, TuJ1, and GFAP confirmed the morphological observations.

Multiple growth factors: Following one week's exposure to a variety ofneural differentiation agents, cells were stained for markers indicativeof neural progenitors (human nestin), neurons (TuJ1), and astrocytes(GFAP). Cells grown in the first stage in non-serum containing media haddifferent morphologies than those cells in serum containing (2% or 10%)media, indicating potential neural differentiation. Specifically,following a two step procedure of exposing umbilicus-derived cells toEGF and bFGF, followed by SHH, FGF8, GDNF, BDNF, and retinoic acid,cells showed long extended processes similar to the morphology ofcultured astrocytes. When 2% FBS or 10% FBS was included in the firststage of differentiation, cell number was increased and cell morphologywas unchanged from control cultures at high density. Potential neuraldifferentiation was not evidenced by immunocytochemical analysis forhuman nestin, TuJ1, or GFAP.

Neural progenitor and PPDC co-culture: PPDCs were plated onto culturesof rat neural progenitors seeded two days earlier in neural expansionconditions (NPE+F+E). While visual confirmation of plated PPDCs provedthat these cells were plated as single cells, human-specific nuclearstaining (hNuc) 4 days post-plating (6 days total) showed that theytended to ball up and avoid contact with the neural progenitors.Furthermore, where PPDCs attached, these cells spread out and appearedto be innervated by differentiated neurons that were of rat origin,suggesting that the PPDCs may have differentiated into muscle cells.This observation was based upon morphology under phase contrastmicroscopy. Another observation was that typically large cell bodies(larger than neural progenitors) possessed morphologies resemblingneural progenitors, with thin processes spanning out in multipledirections. HNuc staining (found in one half of the cell's nucleus)suggested that in some cases these human cells may have fused with ratprogenitors and assumed their phenotype. Control wells containing onlyneural progenitors had fewer total progenitors and apparentdifferentiated cells than did co-culture wells containing umbilicus orplacenta PPDCs, further indicating that both umbilicus- andplacenta-derived cells influenced the differentiation and behavior ofneural progenitors, either by release of chemokines and cytokines, or bycontact-mediated effects.

Summary: Multiple protocols were conducted to determine the short termpotential of PPDCs to differentiate into neural lineage cells. Theseincluded phase contrast imaging of morphology in combination withimmunocytochemistry for nestin, TuJ1, and GFAP, proteins associated withmultipotent neural stem and progenitor cells, immature and matureneurons, and astrocytes, respectively. Evidence was observed to suggestthat neural differentiation occurred in certain instances in theseshort-term protocols.

Several notable observations were made in co-cultures of PPDCs withneural progenitors. This approach, using human PPDCs along with axenogeneic cell type allowed for absolute determination of the origin ofeach cell in these cultures. First, some cells were observed in thesecultures where the cell cytoplasm was enlarged, with neurite-likeprocesses extending away from the cell body, yet only half of the bodylabeled with hNuc protein. Those cells may have been human PPDCs thathad differentiated into neural lineage cells or they may have been PPDCsthat had fused with neural progenitors. Second, it appeared that neuralprogenitors extended neurites to PPDCs in a way that indicates theprogenitors differentiated into neurons and innervated the PPDCs. Third,cultures of neural progenitors and PPDCs had more cells of rat originand larger amounts of differentiation than control cultures of neuralprogenitors alone, further indicating that plated PPDCs provided solublefactors and or contact-dependent mechanisms that stimulated neuralprogenitor survival, proliferation, and/or differentiation.

EXAMPLE 14 Long-Term Neural Differentiation of Postpartum-Derived Cells

The ability of umbilicus and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) to undergo long-term differentiationinto neural lineage cells was evaluated.

Methods & Materials

Isolation and Expansion of PPDCs: PPDCs were isolated and expanded asdescribed in previous Examples.

PPDC Cell Thaw and Plating: Frozen aliquots of PPDCs (umbilicus (022803)P11; (042203) P11; (071003) P12; placenta (101503) P7) previously grownin Growth Medium were thawed and plated at 5,000 cells/cm² in T-75flasks coated with laminin (BD, Franklin Lakes, N.J.) in Neurobasal-Amedium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement,Invitrogen), L-glutamine (4 mM), and Penicillin/Streptomycin (10milliliters), the combination of which is herein referred to as NeuralProgenitor Expansion (NPE) media. NPE media was further supplementedwith bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF(20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

Control Cell Plating: In addition, adult human dermal fibroblasts (P11,Cambrex, Walkersville, Md.) and mesenchymal stem cells (P5, Cambrex)were thawed and plated at the same cell seeding density onlaminin-coated T-75 flasks in NPE+bFGF+EGF. As a further control,fibroblasts, umbilicus, and placenta PPDCs were grown in Growth Mediumfor the period specified for all cultures.

Cell Expansion: Media from all cultures were replaced with fresh mediaonce a week and cells observed for expansion. In general, each culturewas passaged one time over a period of one month because of limitedgrowth in NPE+bFGF+EGF.

Immunocytochemistry: After a period of one month, all flasks were fixedwith cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at roomtemperature. Immunocytochemistry was performed using antibodies directedagainst TuJ1 (BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) and GFAP(glial fibrillary acidic protein; 1:2000; DakoCytomation, Carpinteria,Calif.). Briefly, cultures were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 30 minutes to access intracellular antigens.Primary antibodies, diluted in blocking solution, were then applied tothe cultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing block along with goat anti-mouse IgG—Texas Red (1:250;Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488(1:250; Molecular Probes). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

NPE+bFGF+EGF media slows proliferation of PPDCs and alters theirmorphology Immediately following plating, a subset of PPDCs attached tothe culture flasks coated with laminin. This may have been due to celldeath as a function of the freeze/thaw process or because of the newgrowth conditions. Cells that did attach adopted morphologies differentfrom those observed in Growth Media.

Upon confluence, cultures were passaged and observed for growth. Verylittle expansion took place of those cells that survived passage. Atthis point, very small cells with no spread morphology and withphase-bright characteristics began to appear in cultures ofumbilicus-derived cells. These areas of the flask were followed overtime. From these small cells, bifurcating processes emerged withvaricosities along their lengths, features very similar to previouslydescribed PSA-NCAM+neuronal progenitors and TuJ1+immature neuronsderived from brain and spinal cord (1, 2). With time, these cells becamemore numerous, yet still were only found in clones.

Clones of umbilicus-derived cells express neuronal proteins: Cultureswere fixed at one month post-thawing/plating and stained for theneuronal protein TuJ1 and GFAP, an intermediate filament found inastrocytes. While all control cultures grown in Growth Medium and humanfibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to beTuJ1−/GFAP−, TuJ1 was detected in the umbilicus and placenta PPDCs.Expression was observed in cells with and without neuronal-likemorphologies. No expression of GFAP was observed in either culture. Thepercentage of cells expressing TuJ1 with neuronal-like morphologies wasless than or equal to 1% of the total population (n=3 umbilicus-derivedcell isolates tested). While not quantified, the percentage of TuJ1+cells without neuronal morphologies was higher in umbilicus-derived cellcultures than placenta-derived cell cultures. These results appearedspecific as age-matched controls in Growth Medium did not express TuJ1.

Summary: Methods for generating differentiated neurons (based on TuJ1expression and neuronal morphology) from umbilicus-derived cells weredeveloped. While expression for TuJ1 was not examined earlier than onemonth in vitro, it is clear that at least a small population ofumbilicus-derived cells can give rise to neurons either through defaultdifferentiation or through long-term induction following one month'sexposure to a minimal media supplemented with L-glutamine, basic FGF,and EGF.

EXAMPLE 15 PPDC Trophic Factors for Neural Progenitor Support

The influence of umbilicus- and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) on adult neural stem and progenitorcell survival and differentiation through non-contact dependent(trophic) mechanisms was examined.

Methods & Materials

Adult neural stem and progenitor cell isolation: Fisher 344 adult ratswere sacrificed by CO₂ asphyxiation followed by cervical dislocation.Whole brains were removed intact using bone rongeurs and hippocampustissue dissected based on coronal incisions posterior to the motor andsomatosensory regions of the brain (Paxinos, G. & Watson, C. 1997. TheRat Brain in Stereotaxic Coordinates). Tissue was washed in Neurobasal-Amedium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement;Invitrogen), L-glutamine (4 mM; Invitrogen), and penicillin/streptomycin(Invitrogen), the combination of which is herein referred to as NeuralProgenitor Expansion (NPE) medium. NPE medium was further supplementedwith bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF(20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNase (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/media was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250 g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micrometer cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC plating: Postpartum-derived cells (umbilicus (022803) P12, (042103)P12, (071003) P12; placenta (042203) P12) previously grown in GrowthMedium were plated at 5,000 cells/transwell insert (sized for 24 wellplate) and grown for a period of one week in Growth Medium in inserts toachieve confluence.

Adult neural progenitor plating: Neural progenitors, grown asneurospheres or as single cells, were seeded onto laminin-coated 24 wellplates at an approximate density of 2,000 cells/well in NPE+bFGF+EGF fora period of one day to promote cellular attachment. One day later,transwell inserts containing postpartum cells were added according tothe following scheme:

-   -   a. Transwell (umbilicus-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   b. Transwell (placenta-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   c. Transwell (adult human dermal fibroblasts [1 F1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 microliters)+neural        progenitors (NPE+bFGF+EGF, 1 milliliter)    -   d. Control: neural progenitors alone (NPE+bFGF+EGF, 1        milliliter)    -   e. Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry: After 7 days in co-culture, all conditions werefixed with cold 4% (w/v) paraformaldehyde (Sigma) for a period of 10minutes at room temperature Immunocytochemistry was performed usingantibodies directed against the epitopes listed in Table 15-1. Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop® software (Adobe, San Jose,Calif.).

Quantitative analysis of neural progenitor differentiation:Quantification of hippocampal neural progenitor differentiation wasexamined A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass spectrometry analysis & 2D gel electrophoresis: In order toidentify unique, secreted factors as a result of co-culture, conditionedmedia samples taken prior to culture fixation were frozen down at −80°C. overnight. Samples were then applied to ultrafiltration spin devices(MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

PPDC co-culture stimulates adult neural progenitor differentiation:Following culture with umbilicus- or placenta-derived cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. While both umbilicus- andplacenta-derived cells induced cell differentiation, the degree ofdifferentiation for all three lineages was less in co-cultures withplacenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified (Table 15-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs. 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+ astrocytes andTuJ1+ neurons in culture (47.2% and 8.7% respectively). These resultswere confirmed by nestin staining indicating that progenitor status waslost following co-culture (13.4% vs. 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did however,appear to enhance the survival of neural progenitors.

Identification of unique compounds: Conditioned media from umbilicus-and placenta-derived co-cultures, along with the appropriate controls(NPE media+1.7% serum, media from co-culture with fibroblasts), wereexamined for differences. Potentially unique compounds were identifiedand excised from their respective 2D gels.

Summary: Co-culture of adult neural progenitor cells with umbilicus orplacenta PPDCs results in differentiation of those cells. Resultspresented in this example indicate that the differentiation of adultneural progenitor cells following co-culture with umbilicus-derivedcells is particularly profound. Specifically, a significant percentageof mature oligodendrocytes was generated in co-cultures ofumbilicus-derived cells. In view of the lack of contact between theumbilicus-derived cells and the neural progenitors, this result appearsto be a function of soluble factors released from the umbilicus-derivedcells (trophic effect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H. et al. 2002. Nature417: 39-44).

EXAMPLE 16 Transplantation of Postpartum-Derived Cells

Cells derived from the postpartum umbilicus and placenta are useful forregenerative therapies. The tissue produced by postpartum-derived cells(PPDCs) transplanted into SCID mice with a biodegradable material wasevaluated. The materials evaluated were Vicryl non-woven, 35/65 PCL/PGAfoam, and RAD 16 self-assembling peptide hydrogel.

Methods & Materials

Cell Culture: Placenta- and umbilicus-derived cells were grown in GrowthMedium (DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetalbovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), penicillin/streptomycin(Gibco)) in a gelatin-coated flasks.

Sample Preparation: One million viable cells were seeded in 15microliters Growth Medium onto 5 mm diameter, 2.25 mm thick Vicrylnon-woven scaffolds (64.33 milligrams/cc; Lot#3547-47-1) or 5 mmdiameter 35/65 PCL/PGA foam (Lot#3415-53). Cells were allowed to attachfor two hours before adding more Growth Medium to cover the scaffolds.Cells were grown on scaffolds overnight. Scaffolds without cells werealso incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM;Gibco) immediately before use. The final concentration of cells in RAD16hydrogel was 1×10⁶ cells/100 microliters.

Test Material (N=4/Rx)

-   -   a. Vicryl non-woven+1×10⁶ umbilicus-derived cells    -   b. 35/65 PCL/PGA foam+1×10⁶ umbilicus-derived cells    -   c. RAD 16 self-assembling peptide+1×10⁶ umbilicus-derived cells    -   d. Vicryl non-woven+1×10⁶ placenta-derived cells    -   e. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells    -   f. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells    -   g. 35/65 PCL/PGA foam    -   h. Vicryl non-woven

Animal Preparation: The animals were handled and maintained inaccordance with the current requirements of the Animal Welfare Act.Compliance with the above Public Laws were accomplished by adhering tothe Animal Welfare regulations (9 CFR) and conforming to the currentstandards promulgated in the Guide for the Care and Use of LaboratoryAnimals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 weeks of age: All handling of the SCID mice tookplace under a hood. The mice were individually weighed and anesthetizedwith an intraperitoneal injection of a mixture of 60 milligrams/kgKETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and10 milligrams/kg ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) andsaline. After induction of anesthesia, the entire back of the animalfrom the dorsal cervical area to the dorsal lumbosacral area was clippedfree of hair using electric animal clippers. The area was then scrubbedwith chlorhexidine diacetate, rinsed with alcohol, dried, and paintedwith an aqueous iodophor solution of 1% available iodine. Ophthalmicointment was applied to the eyes to prevent drying of the tissue duringthe anesthetic period.

Subcutaneous Implantation Technique: Four skin incisions, eachapproximately 1.0 cm in length, were made on the dorsum of the mice. Twocranial sites were located transversely over the dorsal lateral thoracicregion, about 5-mm caudal to the palpated inferior edge of the scapula,with one to the left and one to the right of the vertebral column.Another two were placed transversely over the gluteal muscle area at thecaudal sacro-lumbar level, about 5-mm caudal to the palpated iliaccrest, with one on either side of the midline. Implants were randomlyplaced in these sites in accordance with the experimental design. Theskin was separated from the underlying connective tissue to make a smallpocket and the implant placed (or injected for RAD16) about 1-cm caudalto the incision. The appropriate test material was implanted into thesubcutaneous space. The skin incision was closed with metal clips.

Animal Housing: Mice were individually housed in microisolator cagesthroughout the course of the study within a temperature range of 64°F.-79° F. and relative humidity of 30% to 70%, and maintained on anapproximate 12 hour light/12 hour dark cycle. The temperature andrelative humidity were maintained within the stated ranges to thegreatest extent possible. Diet consisted of Irradiated Pico Mouse Chow5058 (Purina Co.) and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology: Excised skin with implant was fixed with 10% neutral bufferedformalin (Richard-Allan Kalamazoo, Mich.). Samples with overlying andadjacent tissue were centrally bisected, paraffin-processed, andembedded on cut surface using routine methods. Five-micron tissuesections were obtained by microtome and stained with hematoxylin andeosin (Poly Scientific Bay Shore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams (without cells)implanted subcutaneously in SCID mice after 30 days. In contrast therewas extensive tissue fill in foams implanted with umbilical-derivedcells or placenta-derived cells. Some tissue ingrowth was observed inVicryl non-woven scaffolds. Non-woven scaffolds seeded with umbilicus-or placenta-derived cells showed increased matrix deposition and matureblood vessels.

Summary: Synthetic absorbable non-woven/foam discs (5.0 mm diameterx1.0mm thick) or self-assembling peptide hydrogel were seeded with eithercells derived from human umbilicus or placenta and implantedsubcutaneously bilaterally in the dorsal spine region of SCID mice. Theresults demonstrated that postpartum-derived cells could dramaticallyincrease good quality tissue formation in biodegradable scaffolds.

EXAMPLE 17 Use of Postpartum-Derived Cells in Nerve Repair

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix®, U.S. Pat. Nos. 5,670,483,5,955,343, US/PCT applications US2002/0160471, WO02/062969) have beendeveloped to act as a scaffold for cell-attachment to encapsulate cellsin 3-D, plate cells in 2-D coatings, or as microcarriers in suspensioncultures. Three-dimensional cell culture has required eitheranimal-derived materials (mouse sarcoma extract), with their inherentreproducibility and cell signaling issues, or much larger syntheticscaffolds, which fail to approximate the physical nanometer-scale andchemical attributes of native ECM. RAD 16 (NH2-(RADA) 3-COOH) and KLD(NH2-(KLDL)3-COOH) are synthesized in small (RAD 16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials

Cells: Cultures of human adult PPDCs (umbilicus and placenta) andfibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with 100 Units per milliliter penicillin, 100 microgramsper milliliter streptomycin, 0.25 micrograms per milliliter amphotericinB (Invitrogen, Carlsbad, Calif.). At passage 11 cells were trypsinizedand viability was determined using trypan blue staining Briefly, 50microliters of cell suspension was combined with 50 microliters of 0.04%w/v trypan blue (Sigma, St. Louis Mo.) and the viable cell number, wasestimated using a hemocytometer. Cells were then washed three times insupplement free-Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.).Cells were then suspended at a concentration of 200,000 cells in 25microliters of RAD-16 (3DM Inc., Cambridge, Mass.), which was bufferedand made isotonic as per manufacturer's recommendations. One hundredmicroliters of supplement free Leibovitz's L-15 medium was added abovethe cell/matrix suspension to keep it wet till use. These cell/matrixcultures were maintained under standard atmospheric conditions untiltransplantation occurred. At the point of transplantation the excessmedium was removed.

Animals and Surgery: Long Evans female rats (220-240 gram body weight)were used. Under intraperitoneal tribromoethanol anesthesia (20milligram/100 grams body weight), the optic nerve was exposed, and theoptic sheath was incised intraorbitally at approximately 2 millimetersfrom the optic disc, the nerve was lifted from the sheath to allowcomplete transsection with fine scissors (Li et al., 2003). Thecompleteness of transsection was confirmed by visually observingcomplete separation of the proximal and distal stumps. The control groupconsisted of lesioned rats without transplants. In transplant ratscultured postpartum cells seeded in RAD-16 were inserted between theproximal and distal stumps using a pair of microforceps. Approximately75,000 cells in RAD-16 were implanted into the severed optic nerve.Cell/matrix was smeared into the severed cut using a pair of finemicroforceps. The severed optic nerve sheath was closed with 10/0 blackmonofilament nylon (Ethicon, Inc., Edinburgh, UK). Thus, the gap wasclosed by drawing the cut proximal and distal ends of the nerve inproximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days post transplantation.

CTB Application: Three days before animals were sacrificed, underanesthesia, a glass micropipette with a 30-50 millimeter tip wasinserted tangentially through the sclera behind the lens, and two 4-5microliter aliquots of a 1% retrograde tracer-cholera toxin B (CTB)aqueous solution (List Biologic, Campbell, Calif.) was injected into thevitreous. Animals were perfused with fixative and optic nerves werecollected in the same fixative for 1 hour. The optic nerves weretransferred into sucrose overnight. Twenty micrometer cryostat sectionswere incubated in 0.1 molar glycine for 30 minutes and blocked in a PBSsolution containing 2.5% bovine serum albumin (BSA) (Boeringer Mannheim,Mannheim, Germany) and 0.5% triton X-100 (Sigma, St. Louis, Mo.),followed by a solution containing goat anti-CTB antibody (List Biologic,Campbell, Calif.) diluted 1:4000 in a PBS containing 2% normal rabbitserum (NRS) (Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% TritonX-100 (Sigma, St. Louis, Mo.) in PBS, and incubated in biotinylatedrabbit anti-goat IgG antibody (Vector Laboratories, Burlinghame, Calif.)diluted 1:200 in 2% Triton-X100 in PBS for 2 hours at room temperature.This was followed by staining in 1:200 streptavidin-green (Alexa Flour438; Molecular Probes, Eugene, Oreg.) in PBS for 2 hours at roomtemperature. Stained sections were then washed in PBS and counterstainedwith propidium iodide for confocal microscopy.

Histology Preparation: Briefly, 5 days after CTB injection, rats wereperfused with 4% paraformaldehyde. Rats were given 4 cubic centimetersof urethane and were then perfused with PBS (0.1 molar) then with 4%Para formaldehyde. The spinal cord was cut and the bone removed from thehead to expose the colliculus. The colliculus was then removed andplaced in 4% paraformaldehyde. The eye was removed by cutting around theoutside of the eye and going as far back as possible. Care was given notto cut the optic nerve that lies on the underside of the eye. The eyewas removed and the muscles were cut exposing the optic nerve this wasthen placed in 4% paraformaldehyde.

Results

Lesions alone: One month after retrotubular section of the optic nerve,a number of CTB-labeled axons were identified in the nerve segmentattached to the retina. In the 200 micrometers nearest the cut, axonswere seen to emit a number of collaterals at right angles to the mainaxis and terminate as a neuromatous tangle at the cut surface. In thiscut between the proximal and distal stumps, the gap was observed to beprogressively bridged by a 2-3 millimeter segment of vascularizedconnective tissue; however, no axons were seen to advance into thisbridged area. Thus, in animals that received lesion alone no axonalgrowth was observed to reach the distal stump.

RAD-16 transplantation: Following transplantation of RAD-16 into thecut, visible ingrowth of vascularized connective tissue was observed.However, no axonal in growth was observed between the proximal anddistal stumps. The results demonstrate that application of RAD-16 aloneis not sufficient for inducing axonal regeneration in this situation.

Transplantation of postpartum-derived cells: Transplantation ofpostpartum-derived cells into the severed optic nerve stimulated opticnerve regrowth. Some regrowth was also observed in conditions in whichfibroblast cells were implanted, although this was minimal as comparedwith the regrowth observed with the transplanted placenta-derived cells.Optic nerve regrowth was observed in ⅘ animals transplanted withplacenta-derived cells, 3/6 animals transplanted with adult dermalfibroblasts and in ¼ animals transplanted with umbilicus-derived cells.In situations where regrowth was observed, CTB labeling confirmedregeneration of retinal ganglion cell axons, which were demonstrated topenetrate through the transplant area. GFAP labeling was also performedto determine the level of glial scarring. The GFAP expression wasintensified at the proximal stump with some immunostaining beingobserved through the reinervated graft.

Summary: These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons.

EXAMPLE 18 Use of Postpartum-Derived Cells in the Treatment of RetinitisPigmentosa

Currently no real treatment exists for blinding disorders that stem fromthe degeneration of cells in the retina. Loss of photoreceptors as aresult of apoptosis or secondary degeneration lead to progressivedeterioration of vision, and ultimately to blindness. Diseases in whichthis occurs include age-related macular degeneration (AMD) and retinitispigmentosa (RP). RP is most commonly associated with a single genemutation, which contributes to photoreceptor cell death.

The retinal photoreceptors and adjacent retinal pigment epithelium forma functional unit. The Royal College of Surgeons (RCS) rat presents witha tyrosine receptor kinase (Merkt) defect affecting outer segmentphagocytosis, leading to photoreceptor cell death. Transplantation ofretinal pigment epithelial (RPE) cells into the subretinal space of RCSrats was found to limit the progress of photoreceptor loss and preservevisual function. In this example, it is demonstrated thatpostpartum-derived cells can be used to promote photoreceptor rescue andthus preserve photoreceptors in an RCS model.

Methods & Materials

Cell transplants: Cultures of human adult umbilicus-derived cells,placental-derived cells and fibroblast cells (passage 10) were expandedfor 1 passage. All cells were initially seeded at 5,000 cells/cm² ongelatin-coated T75 flasks in Growth Medium. For subsequent passages, allcells were treated as follows. After trypsinization, viable cells werecounted after trypan blue staining Briefly, 50 microliters of cellsuspension was combined with 50 microliters of 0.04% w/v trypan blue(Sigma, St. Louis Mo.) and the viable cell number was estimated using ahemocytometer. Cells were trypsinized and washed three times insupplement free-DMEM: Low glucose medium (Invitrogen, Carlsbad, Calif.).Cultures of human adult umbilicus-derived cells, placental-derived cellsand fibroblast cells at passage 11 were trypsinized and washed twice inLeibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.). For thetransplantation procedure, dystrophic RCS rats were anesthetized withxylazine-ketamine (1 mg/kg i.p. of the following mixture: 2.5 mlxylazine at 20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml distilledwater) and their heads secured by a nose bar. Cells devoid of serum wereresuspended (2×10⁵ cells per injection) in 2 microliters of Leibovitz,L-15 medium (Invitrogen, Carlsbad, Calif.) and transplanted using a fineglass pipette (internal diameter 75-150 micrometers) trans-scerally.Cells were delivered into the dorso-temporal subretinal space ofanesthetized 3 week old dystrophic-pigmented RCS rats (total N=10/celltype).

Cells were injected unilaterally into the right eye, while the left eyewas injected with carrier medium alone (Sham control; Leibovitz's L-15medium). Viability of residual transplant cells remained at greater than95% as assessed by trypan blue exclusion at the end of the transplantsession. After cell injections were performed, animals were injectedwith dexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at 60 or 90 dayspostoperatively, with some animals being sacrificed at earliertimepoints for histological assessment of short-term changes associatedwith cell transplantation.

ERG recordings: Following overnight dark adaptation, animals wereprepared for ERG recording under dim red light, as described in (Sauve,Y. et al., 2004, Vision Res. 44: 9-18). In brief, under anesthesia (witha mixture of 150 mg/kg i.p. ketamine, and 10 mg/kg i.p. xylazine) thehead was secured with a stereotaxic head holder and the body temperaturemonitored through a rectal thermometer and maintained at 38° C. using ahomeothermic blanket. Pupils were dilated using equal parts of topical2.5% phenylephrine and 1% tropicamide. Topical anesthesia with 0.75%bupivacaine was used to prevent any corneal reflexes and a drop of 0.9%saline was frequently applied on the cornea to prevent its dehydrationand allow electrical contact with the recording electrode (gold wireloop). A 25-gauge needle inserted under the scalp, between the two eyes,served as the reference electrode. Amplification (at 1-1000 Hz bandpass,without notch filtering), stimulus presentation, and data acquisitionwere provided by the UTAS-3000 system from LKC Technologies(Gaithersburg, Md.). ERGs were recorded at 60 and 90 days of age in theumbilicus-derived cell groups and at 60 days only in theplacental-derived cell and fibroblast cell groups.

Mixed a- and b-wave recording, measuring total rod and conecontribution: For the quantification of dark-adapted b-waves, recordingsconsisted of single flash presentations (10 μsec duration), repeated 3to 5 times to verify the response reliability and improve thesignal-to-noise ratio, if required. Stimuli were presented at sixincreasing intensities in one log unit steps varying from −3.6 to 1.4log candila/m² in luminance. To minimize the potential bleaching ofrods, inter-stimulus intervals were increased as the stimulus luminancewas elevated from 10 sec at lowest stimulus intensity to 2 minutes athighest stimulus intensity. The maximum b-wave amplitude was defined asthat obtained from the flash intensity series, regardless of thestimulus intensity. The true V_(max) from fitting the data with aNaka-Rushton curve was not used because ERG responses were often erraticat higher luminance levels in dystrophic animals and showed tendenciesfor depressed responses around 0.4 and 1.4 log candila/m². In order todetermine the age at which ERG components were obtained or lost,criterion amplitudes were used: 20 μV for a- and b-waves, and 10 μV forSTR-like responses. The amplitude of the b-wave was measured from thea-wave negative peak up to the b-wave positive apex, and not up to thepeak of oscillations, which can exceed the b-wave apex. In theseexperiments as disease progresses in this model the ERG's areeffectively abnormal. Thus, ERG measurements are taken on the lower endof normal visual function. This in turn can make measurements noisy asyou reach the limits of threshold sensitivity.

Isolation of rod and cone responses: The double flash protocol, asdescribed in (Nixon, P. J. et al., 2001, Clin Experiment Opthalmol.29:193-196) was used to determine the isolation of rod and coneresponses. A probe flash was presented 1 sec after a conditioning flash,using a specific feature of the UTAS-3000 system (LKC Technologies) withcalibrated ganzfeld; assuring complete recharge of the stimulator underthe conditions used. The role of the conditioning flash in the procedurewas to transiently saturate rods so that they were rendered unresponsiveto the probe flash. Response to the probe flash was taken as reflectingcone-driven activity. A rod-driven b-wave was obtained by subtractingthe cone-driven response from the mixed response (obtained followingpresentation of a probe flash alone, i.e. not preceded by anyconditioning flash).

Functional Assessment: Physiological retinal sensitivity testing wasperformed to demonstrate retinal response to dim light. Animals wereanesthetized with a recovery dose of urethane at 1.25 g/kg i.p.Physiological assessment in the animals was tested post graft in animalsat 90 days by recording multiunit extracellular activity in the superiorcolliculus to illumination of respective visual receptive fields, usingthe method disclosed in (Lund, R. D. et al., 2001, Proc. Natl. Acad.Sci. USA. 98: 9942-9947). This procedure was repeated for 20 independentpoints (spaced 200 mm apart, with each step corresponding toapproximately 10-150 displacements in the visual field), covering thevisual field. Visual thresholds were measured as the increase inintensity over background and maintained at 0. 02 candila/m²(luminescence unit) [at least 2.6 logarithm units below rod saturation],required for activating units in the superficial 200 μm of the superiorcolliculus. Response parameters were compared between transplanted andsham control eyes that received vehicle alone.

Histology: Animals were sacrificed with an overdose of urethane (12.5g/kg). The orientation of the eye was maintained by placing a 6.0 suturethrough the superior rectus muscle prior to enucleation. After making acorneal incision, the eyes were fixed with 2.5% parafomaldehyde, 2.5%glutaraldehyde, 0.01% picric acid in 0.1 M cacodylate buffer (pH7.4)(Sigma, St. Louis, Mo.). After fixation, the cornea and lens wereremoved by cutting around the ciliary body. A small nick was made in theperiphery of the dorsal retina prior to removal of the superior rectusto assist in maintaining orientation. The retinas were then post-fixedin 1% osmium tetroxide for 1 h. After dehydration through a series ofalcohols to epoxypropane, the retinas were embedded in TAAB embeddingresin (TAAB Laboratories, Aldemarston, UK). Semi-thin sections werestained with 1% toluidine Blue in 1% borate buffer and the ultra thinsections were contrasted with uranyl acetate and lead citrate.

For Nissl staining, sections were stained with 0.75% cresyl violet(Sigma, St. Louis, Mo.) after which they were dehydrated through gradedalcohols at 70, 95 and 100% twice. They were then placed in xylene(Sigma, St. Louis, Mo.), rinsed with PBS (pH 7.4) (Invitrogen, Carlsbad,Calif.), coverslipped and mounted with DPX mountant (Sigma, St. Louis,Mo.).

Results

ERG Recordings: Animals that received umbilicus-derived cell injectionsexhibited relative preservation of visual response properties 60 and 90days post-operatively (Table 18-1). The response observed in theseanimals was greater than that seen with placental-derived cell,fibroblast cell or sham treated animals. Placental-derived celltransplants (n=4) at 60 days showed no improvement in a-wave (20±20)versus sham controls (O), but showed some improvement in mixed b-wave(81±72) versus sham controls (1.5±2), and good improvement incone-b-wave (50±19) versus sham controls (7±7), and in rod contribution(30%) versus sham controls (O). These results indicated some improvementin visual responsiveness when compared to sham controls.

Umbilicus-derived cell-transplanted animals (n=6) demonstrated goodimprovement in all outcome measures tested at 60 days (Table 18-1),a-wave (27±11) versus sham controls (O), mixed b-wave (117±67) versussham controls (18±13), cone-b-wave (55±25) versus sham controls (28±11),and in rod contribution (49±16%) versus sham controls (6±7%).Furthermore, at 90 days, improved responses were measured in two animalstested, with measures including: a-wave (15±7) versus sham controls (O),mixed b-wave (37±15) versus sham controls (O), cone-b-wave (16±11)versus sham controls (7±5), and in rod contribution (58±39%) versus shamcontrols (0%). These results indicate that visual responsiveness wasimproved in umbilicus-derived cell transplanted animals with evidencefor photoreceptor preservation in the RCS model. Although a diminutionin responsiveness to ERG was observed in the 90 day animals tested,their preservation of visual function in comparison to sham-treatedcontrols was good.

In contrast to either umbilicus-derived or placental-derived cells,fibroblast transplantations showed no improvement in any of theparameters tested, with values less than or equal to sham-treatedcontrols.

Histology: Following transplantation, there was no histological evidenceof an inflammatory reaction and infiltrating immune cells were notobserved in Nissl-stained sections in the postpartum cell groups.However, fibroblast implantations resulted in animal death (n=7) andindications of early stage inflammatory responses. Histologically at the90 day time point in the umbilicus-derived cell transplanted animalsanatomical rescue of photoreceptors was clearly demonstrated. Thephotoreceptors formed a thick layer separated by a gap from the innernuclear layer, made up of other retinal cells. By comparison, the widthof the outer layer in the sham control was, at best, a discontinuoussingle layer as opposed to around 5 cells thick in the grafted eye. Incomparison to a normal animal this is marginally more than half thethickness of photoreceptor cell layers normally observed.

Functional Assessment: Efficacy of transplants in preventing visual losswas monitored by assessment of electrophysiological responsiveness intwo animals. The threshold sensitivity response to light was used todefine the area of visual field rescue in sham-injected control eyesversus eyes transplanted with umbilicus-derived cells. In nondystrophicrats, visual thresholds never exceeded 0.5 log candila/m² abovebackground. In non-operated dystrophic rats, the thresholds are usuallyin the magnitude of 4 log candila/m² units. By contrast, in non-operatedsham injected dystrophic rats, the thresholds were in the order of2.9-4.9 log candila/m² units with an average threshold of 4.0 logcandila/m² units, in some instances no recording could be attained.Thus, the sham-injected rats showed some highly localized functionalrescue in the temporal retina. However, the human umbilicus-derived celltransplanted rats exhibited substantially greater levels of visualpreservation with thresholds ranging from 0.8 to 2.1 log candila/m²units, with an average threshold of 1.3 log candila/m² units.

Summary: Transplantation of umbilicus-derived cells into dystrophic RCSrats was shown to preserve photoreceptors. To a lesser degree thisresponse was also seen following transplantation with placental-derivedcells although improvement was seen in a-wave responsiveness was notobserved. In this degenerative model, one would expect the a-wave todisappear within 30 to 60 days and the b-wave to disappear within 3months. Thus, a retained a-wave indicates that real and normal rodfunction is preserved. Rod contribution to b-wave suggests abnormal rodfunction is still possible. The sustained non-rod b-wave is the measureof how much cone function is maintained, which is a real measure ofvision. Thus, the level of improvement assessed both physiologically andanatomically following umbilicus-derived cell transplantation is welldefined here. ERG measurements provide an assessment of visual functionafter photoreceptor loss, indicating changes in electrical activity inthe retina. However, ERG does not provide direct information as to imageforming capability. The measurement of collicular threshold sensitivityused in this study provides an indication of relative preservation ofvisual fields. The importance of this measure is based on a correlationbetween the amounts of functional rescue and anatomical preservation andthat the data collected compares with visual field perimetry testing inhumans. The transplantation has demonstrated a retardation of thedisease process in the test animals. Thus, the results presented hereindemonstrate clear evidence of functional efficacy of grafting humanPPDCs into the subretinal space, and that rescue occurs in the generalregion in which the grafted cells are located.

EXAMPLE 19 Use of Postpartum Derived Cells in the Treatment of Glaucoma

Glaucoma is a group of eye diseases causing optic nerve damage. Theoptic nerve carries images from the retina, which is the specializedlight sensing tissue, to the brain so we can see. In glaucoma, eyepressure plays a role in damaging the delicate nerve fibers of the opticnerve. When a significant number of nerve fibers are damaged, blindspots develop in the field of vision. Once nerve damage and visual lossoccur, it is permanent. Most people don't notice these blind areas untilmuch of the optic nerve damage has already occurred. If the entire nerveis destroyed, blindness results. Furthermore, this damage to the opticnerve leads to damage of retinal ganglion cell elements, whichcorrespondingly lead to loss of photoreceptors and a subsequent loss ofvision. Glaucoma is a leading cause of blindness in the world,especially in older people. Thus, if a therapy can be provided, that caneither regenerate new nerve fibers or replace existing ones, thepotential to repair the degenerative process in Glaucoma exists. In thefollowing example we describe the potential of umbilicus-derived cellsto stimulate the generation of neurons and oligodendrocytes. Thegeneration of these cell types in vivo would enable replacement of lostnerve fibers.

Methods & Materials

Adult neural stem and progenitor cell isolation: Fisher adult rats weresacrificed by CO₂ asphyxiation followed by cervical dislocation. Wholebrains were removed intact using bone rongeurs and hippocampus tissuedissected based on coronal incisions posterior to the motor andsomatosensory regions of the brain (Paxinos, G. & Watson, C. 1997. TheRat Brain in Stereotaxic Coordinates). Tissue was washed in Neurobasal-Amedium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement;Invitrogen), L-glutamine (4 mM; Invitrogen), and penicillin/streptomycin(Invitrogen), the combination of which is herein referred to as NeuralProgenitor Expansion (NPE) medium. NPE medium was further supplementedwith bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF(20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/media was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250 g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micrometer cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC plating: Postpartum-derived cells (umbilicus (022803) P12, (042103)P12, (071003) P12; placenta (042203) P12) previously grown in GrowthMedium were plated at 5,000 cells/transwell insert (sized for 24 wellplate) and grown for a period of one week in Growth Medium in inserts toachieve confluence.

Adult neural progenitor plating: Neural progenitors, grown asneurospheres or as single cells, were seeded onto laminin-coated 24 wellplates at an approximate density of 2,000 cells/well in NPE+bFGF+EGF fora period of one day to promote cellular attachment. One day later,transwell inserts containing postpartum cells were added according tothe following scheme:

-   -   1. Transwell (umbilicus-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   2. Transwell (placenta-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   3. Transwell (adult human dermal fibroblasts [1 F1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 microliters)+neural        progenitors (NPE+bFGF+EGF, 1 milliliter)    -   4. Control: neural progenitors alone (NPE+bFGF+EGF, 1        milliliter)    -   5. Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry: After 7 days in co-culture, all conditions werefixed with cold 4% (w/v) paraformaldehyde (Sigma) for a period of 10minutes at room temperature Immunocytochemistry was performed usingantibodies directed against the epitopes listed in Table 19-1. Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color video camera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Quantitative analysis of neural progenitor differentiation:Quantification of hippocampal neural progenitor differentiation wasexamined A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass spectrometry analysis & 2D gel electrophoresis: In order toidentify unique, secreted factors as a result of co-culture, conditionedmedia samples taken prior to culture fixation were frozen down at −80°C. overnight. Samples were then applied to ultrafiltration spin devices(MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

PPDC co-culture stimulates adult neural progenitor differentiation.Following culture with umbilicus- or placenta-derived cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. While both umbilicus- andplacenta-derived cells induced cell differentiation, the degree ofdifferentiation for all three lineages was less in co-cultures withplacenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified (Table 19-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs. 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+ astrocytes andTuJ1+ neurons in culture (47.2% and 8.7% respectively). These resultswere confirmed by nestin staining indicating that progenitor status waslost following co-culture (13.4% vs. 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did, however,appear to enhance the survival of neural progenitors.

Identification of unique compounds: Conditioned media from umbilicus-and placenta-derived co-cultures, along with the appropriate controls(NPE media±1.7% serum, media from co-culture with fibroblasts), wereexamined for differences. Potentially unique compounds were identifiedand excised from their respective 2D gels.

Summary: Co-culture of adult neural progenitor cells with umbilicus orplacenta PPDCs results in differentiation of those cells. Resultspresented in this example indicate that the differentiation of adultneural progenitor cells following co-culture with umbilicus-derivedcells is particularly profound. Specifically, a significant percentageof mature oligodendrocytes was generated in co-cultures ofumbilicus-derived cells. In view of the lack of contact between theumbilicus-derived cells and the neural progenitors, this result appearsto be a function of soluble factors released from the umbilicus-derivedcells (trophic effect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H. et al. 2002. Nature417: 39-44).

The demonstration that postpartum cells derived from umbilicus tissuecan promote the differentiation of neural stem cells into neurons andoligodendrocytes suggests that the cells may promote axonal regeneration(see Example 20) or remyelination. These data suggest that postpartumcells derived from umbilicus tissue may be protective in glaucoma.Furthermore, if postpartum cells derived from umbilicus tissue areinjected in combination with neural stem cells they would have thepotential to remyelinate lost neural elements in the retina, whichsupport vision.

EXAMPLE 20 Use of Postpartum-Derived Cells in Optic Nerve Repair for theTreatment of Glaucoma

Glaucoma is a group of eye diseases causing optic nerve damage. Theoptic nerve carries images from the retina, which is the specializedlight sensing tissue, to the brain so we can see. In glaucoma, eyepressure plays a role in damaging the delicate nerve fibers of the opticnerve. When a significant number of nerve fibers are damaged, blindspots develop in the field of vision. Once nerve damage and visual lossoccur, it is permanent. Most people don't notice these blind areas untilmuch of the optic nerve damage has already occurred. If the entire nerveis destroyed, blindness results. Glaucoma is a leading cause ofblindness in the world, especially in older people. Thus, if you areable to provide a therapy that can either regenerate new nerve fibers orreplace existing ones, the potential to repair the degenerative processin Glaucoma exists. The following Example demonstrates the ability ofumbilicus-derived cells to cause axonal re-growth through a severedoptic nerve head. Such growth in vivo could provide the level of axonalregeneration required to repair vision loss.

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix®, U.S. Pat. Nos. 5,670,483,5,955,343, US/PCT applications US2002/0160471, WO02/062969) have beendeveloped to act as a scaffold for cell-attachment to encapsulate cellsin 3-D, plate cells in 2-D coatings, or as microcarriers in suspensioncultures. Three-dimensional cell culture has required eitheranimal-derived materials (mouse sarcoma extract), with their inherentreproducibility and cell signaling issues, or much larger syntheticscaffolds, which fail to approximate the physical nanometer-scale andchemical attributes of native ECM. RAD 16 (NH2-(RADA) 3-COOH) and KLD(NH2-(KLDL)3-COOH) are synthesized in small (RAD 16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials

Cells. Cultures of human adult PPDCs (umbilicus and placenta) andfibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with 100 Units per milliliter penicillin, 100 microgramsper milliliter streptomycin, 0. 25 micrograms per milliliteramphotericin B (Invitrogen, Carlsbad, Calif.). At passage 11 cells weretrypsinized and viability was determined using trypan blue stainingBriefly, 50 microliters of cell suspension was combined with 50microliters of 0.04% w/v trypan blue (Sigma, St. Louis Mo.) and theviable cell number, was estimated using a hemocytometer. Cells were thenwashed three times in supplement free-Leibovitz's L-15 medium(Invitrogen, Carlsbad, Calif.). Cells were then suspended at aconcentration of 200,000 cells in 25 microliters of RAD-16 (3DM Inc.,Cambridge, Mass.), which was buffered and made isotonic as permanufacturer's recommendations. One hundred microliters of supplementfree Leibovitz's L-15 medium was added above the cell/matrix suspensionto keep it wet till use. These cell/matrix cultures were maintainedunder standard atmospheric conditions until transplantation occurred. Atthe point of transplantation the excess medium was removed.

Animals and Surgery: Long Evans female rats (220-240 gram body weight)were used. Under intraperitoneal tribromoethanol anesthesia (20milligram/100 grams body weight), the optic nerve was exposed, and theoptic sheath was incised intraorbitally at approximately 2 millimetersfrom the optic disc, the nerve was lifted from the sheath to allowcomplete transsection with fine scissors (Li et al., 2003). Thecompleteness of transsection was confirmed by visually observingcomplete separation of the proximal and distal stumps. The control groupconsisted of lesioned rats without transplants. In transplant ratscultured postpartum cells seeded in RAD-16 were inserted between theproximal and distal stumps using a pair of microforceps. Approximately75,000 cells in RAD-16 were implanted into the severed optic nerve.Cell/matrix was smeared into the severed cut using a pair of finemicroforceps. The severed optic nerve sheath was closed with 10/0 blackmonofilament nylon (Ethicon, Inc., Edinburgh, UK). Thus, the gap wasclosed by drawing the cut proximal and distal ends of the nerve inproximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days post transplantation.

CTB Application: Three days before animals were sacrificed, underanesthesia, a glass micropipette with a 30-50 millimeter tip wasinserted tangentially through the sclera behind the lens, and two 4-5microliter aliquots of a 1% retrograde tracer-cholera toxin B (CTB)aqueous solution (List Biologic, Campbell, Calif.) was injected into thevitreous. Animals were perfused with fixative and optic nerves werecollected in the same fixative for 1 hour. The optic nerves weretransferred into sucrose overnight. Twenty micrometer cryostat sectionswere incubated in 0.1 molar glycine for 30 minutes and blocked in a PBSsolution containing 2.5% bovine serum albumin (BSA) (Boeringer Mannheim,Mannheim, Germany) and 0.5% triton X-100 (Sigma, St. Louis, Mo.),followed by a solution containing goat anti-CTB antibody (List Biologic,Campbell, Calif.) diluted 1:4000 in a PBS containing 2% normal rabbitserum (NRS) (Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% TritonX-100 (Sigma, St. Louis, Mo.) in PBS, and incubated in biotinylatedrabbit anti-goat IgG antibody (Vector Laboratories, Burlinghame, Calif.)diluted 1:200 in 2% Triton-X100 in PBS for 2 hours at room temperature.This was followed by staining in 1:200 streptavidin-green (Alexa Flour438; Molecular Probes, Eugene, Oreg.) in PBS for 2 hours at roomtemperature. Stained sections were then washed in PBS and counterstainedwith propidium iodide for confocal microscopy.

Histology Preparation: Briefly, 5 days after CTB injection, rats wereperfused with 4% paraformaldehyde. Rats were given 4 cubic centimetersof urethane and were then perfused with PBS (0.1 molar) then with 4%Para formaldehyde. The spinal cord was cut and the bone removed from thehead to expose the colliculus. The colliculus was then removed andplaced in 4% paraformaldehyde. The eye was removed by cutting around theoutside of the eye and going as far back as possible. Care was given notto cut the optic nerve that lies on the underside of the eye. The eyewas removed and the muscles were cut exposing the optic nerve this wasthen placed in 4% paraformaldehyde.

Results

Lesions alone: One month after retrotubular section of the optic nerve,a number of CTB-labeled axons were identified in the nerve segmentattached to the retina. In the 200 micrometers nearest the cut, axonswere seen to emit a number of collaterals at right angles to the mainaxis and terminate as a neuromatous tangle at the cut surface. In thiscut between the proximal and distal stumps, the gap was observed to beprogressively bridged by a 2-3 millimeter segment of vascularizedconnective tissue; however, no axons were seen to advance into thisbridged area. Thus, in animals that received lesion alone no axonalgrowth was observed to reach the distal stump.

RAD-16 transplantation: Following transplantation of RAD-16 into thecut, visible ingrowth of vascularized connective tissue was observed.However, no axonal in growth was observed between the proximal anddistal stumps. The results demonstrate that application of RAD-16 aloneis not sufficient for inducing axonal regeneration in this situation.

Transplantation of postpartum-derived cells: Transplantation ofpostpartum-derived cells into the severed optic nerve stimulated opticnerve regrowth. Some regrowth was also observed in conditions in whichfibroblast cells were implanted, although this was minimal as comparedwith the regrowth observed with the transplanted placenta-derived cells.Optic nerve regrowth was observed in ⅘ animals transplanted withplacenta-derived cells, 3/6 animals transplanted with adult dermalfibroblasts and in ¼ animals transplanted with umbilicus-derived cells.In situations where regrowth was observed, CTB labeling confirmedregeneration of retinal ganglion cell axons, which were demonstrated topenetrate through the transplant area. GFAP labeling was also performedto determine the level of glial scarring. The GFAP expression wasintensified at the proximal stump with some immunostaining beingobserved through the reinervated graft.

Summary: These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons. Thus, an ability to promote axonalregeneration from the optic nerve has the potential to repair a primarydefect associated to vision loss with glaucoma.

EXAMPLE 21 Evaluation of the Ability of Umbilicus-Derived Cells toProtect Photoreceptors in Royal College of Surgeons Rat Model of RetinalDegeneration

The central unifying aspect of disease progression in all forms ofretinal degeneration is photoreceptor cell death leading to permanentblindness. With few exceptions such as retinal trauma, this death ismediated through a process termed apoptosis or programmed cell death inall of these diseases.

In order to assess whether application of umbilicus-derived cells couldrescue photoreceptors from apoptosis, umbilicus-derived cells weretransplanted and assessed for their ability to reduce the number ofapoptotic cells in the Royal College of Surgeon's rat model of retinaldegeneration (RCS). In this model, a mutation in retinal pigmentedepithelial (RPE) cells leads to build up of the otherwise phagocytosedouter segments of photoreceptors. As a result of this buildup,photoreceptors become uncoupled from RPE and undergo apoptosis.

One of the characteristics of apoptosis is the degradation of DNA afterthe activation of Ca/Mg dependent endonucleases. This DNA cleavage leadsto strand breakage within the DNA. One accepted measure of detectingapoptosis in histological sections is called TUNEL (terminaldeoxynucleotidyl transferase biotin-dUTP nick end labeling). TUNELidentifies apoptotic cells in situ by using terminal deoxynucleotidyltransferase (TdT) to transfer biotin-dUTP to these strand breaks ofcleaved DNA. The biotin labeled cleavage sites are then detected byreaction with fluorophore-conjugated streptavidin and visualized usingan epifluorescent microscope. In this study, TUNEL labeling was used toassess the percentage of positive apoptotic photoreceptor cells relativeto total nuclei in the outer nuclear layer.

Materials and Methods

Cell transplants: Frozen vials of previously expanded umbilicus-derivedcells (passage 10) were thawed, washed with PBS, and concentrated inDulbecco's Modified Eagles Medium (DMEM; Invitrogen, Grand Island, N.Y.)to 20,000 cells per microliter. Prior to this, cells wereisolated/expanded and cryopreserved in Growth Medium as previouslydescribed in Example 5.

For the transplantation procedure, dystrophic RCS rats were anesthetizedwith xylazine-ketamine (1 mg/kg i.p. of the following mixture: 2.5 mlxylazine at 20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml distilledwater) and their heads secured by a nose bar. Cells were transplantedusing a fine glass pipette (internal diameter 75-150 micrometers)trans-sclerally. Cells were delivered via single injections into thedorso-temporal subretinal space of anesthetized 3 week olddystrophic-pigmented RCS rats (total N=2/group). As controls, sets ofRCS animals underwent either a sham injection procedure containing DMEMonly or were left untreated. Congenic, non-dystrophic rats were includedas a third control group to evaluate the level of normal apoptosis inhealthy eyes.

After cell injections were performed, animals were injected withdexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at 8 days(dystrophic-untreated, dystrophic-sham injected, dystrophic-cellinjected, congenic control) or 67 days postoperatively(dystrophic-untreated, dystrophic-cell injected) for histologicalanalysis.

Histology: Animals were sacrificed with an overdose of urethane (12.5g/kg). The orientation of the eye was maintained by placing a 6.0 suturethrough the superior rectus muscle prior to enucleation. The cornea andlens were next removed by cutting around the ciliary body. Eyes werethen fixed with Davidson's fix containing 22% (v/v) formalin, 33% (v/v)alcohol, 11% (v/v) glacial acetic acid, and 33% (v/v) water. After 24hours, eyes were removed from fix and washed/retained in PBS forsectioning.

Eye samples were oriented, embedded in OCT (Sakura, Torrence, Calif.),and cryostat sectioned (10 μm). TUNEL staining was performed using an insitu apoptosis detection kit whereby fragmented DNA was labeled with TdTand biotin-dUTP. A streptavidin-FITC system was used to visualizelabeling. Sections were counterstained with DAPI (10 μM, Invitrogen) tovisualize cell nuclei and coverslipped.

Imaging & Analysis: Tiled images of TUNEL and DAPI stained retina weretaken at 20× using a scanning stage and TURBOSCAN software (MediaCybernetics Inc, Bethesda, MD). ImagePro Software (Media CyberneticsInc.) was then used to quantitatively analyze the area of DAPI positivenuclei and TUNEL+nuclei in the outer nuclear layer (ONL) aftercalibrating images to square micron area. A minimum of 8 tissue sectionswas analyzed per eye and averaged to generate mean+/−standard deviationvalues per section. TUNEL+area was next divided by DAPI+area to assessthe percentage of nuclei undergoing apoptosis as determined by TUNEL.Post-hoc statistic analysis was performed to assess whether treatmentwith postpartum cells derived from umbilical tissue significantlyreduced the number of TUNEL+figures versus controls (untreated or shaminjected). One-tailed, paired t-tests were performed to evaluate thispossibility.

Results

TUNEL+ area in the photoreceptor specific outer nuclear layer (ONL) wasassessed as a function of DAPI+ area in RCS animals that were untreated,sham injected, or umbilicus-derived cell treated at two differenttimepoints, 8 and 67 days post injection (post natal day 29 and 88respectively). Congenic-untreated controls at P29 were used to assessthe level of apoptosis in healthy retina. Results of this analysis aresummarized in Table 22-1.

At post natal day 29, congenic eyes had very few TUNEL+ figures(0.2±0.2% of total DAPI+ area) in the ONL, while 16.0±2.3% of the ONLwas TUNEL+ in sham injected dystrophic rats. umbilicus-derived celltreated animals, however, exhibited a significant decrease in TUNEL+area (6.6±0.5%, p<0.05, vs. dystrophic-sham). This decrease was not seenin the overall DAPI+ area at this time, suggesting that changes inapoptotic figures had not had an impact on overall photoreceptor numberin the ONL at this timepoint.

In contrast, at P88, there was a significant increase in the DAPI+ areaof untreated vs. umbilicus-derived cell treated animals (p<0.05,dystrophic untreated vs. umbilicus-derived cell) (Table 22-2). Thisdifference was shadowed by differences in the percent of TUNEL+ cells(35.9±9.0% dystrophic untreated vs. 3.8%±1.4% indystrophic-umbilicus-derived cell treated) indicating that at 67 dayspost injection, there was a lasting effect on preservation ofphotoreceptors in umbilicus-derived cell treated animals.

Summary: Decreases in TUNEL+ area, indicative of decreased apoptosis,were observed as early as 8 days post injection and as late as 67 daysfollowing single dose subretinal administration of umbilicus-derivedcells (20,000 cells). This difference is confirmed by an overallsignificant increase in the total area of DAPI+ cells with time inumbilicus-derived cell treated animals, indicating an overallpreservation of photoreceptors as late as post natal day 88. To brieflysummarize, umbilicus-derived cell treatment has an overall positiveeffect in the retina, decreasing the overall TUNEL+ area (and presumablenumber of cells). This effect is lasting and maintained at 67 days postinjection.

EXAMPLE 22 Evaluation of the Ability of Umbilicus-Derived Cells toStimulate RPE Phagocytosis In Vitro

One of the prominent characteristics of retinal pigmented epithelialcells (RPE) is to phagocytose the shed outer segments of photoreceptorson a daily basis. This feature is well characterized and important inmaintaining a healthy retina. However, there are numerous otherfunctions of RPE that are well described and important to maintaininghomeostasis in the eye (reviewed in Strauss et al., 2005). RPEtransportions, water, and metabolic products to the blood, theytransport non-endogenous factors as well as secrete endogenous factorsthat maintain the integrity of photoreceptors as well aschoriocapillaris. RPE also secrete immunosuppressive agents thatestablish immune privilege in the eye. A failure of any one of thesefunctions can lead to degeneration of the retina, loss of visualfunction, and eventually blindness.

There is considerable building evidence to suggest that defects in RPEability to perform these functions can trigger known human diseases likeRetinitis Pigmentosa and the dry form of age-related maculardegeneration (AMD) (Gal et al., 2000; Inana et al., 2005; Inana et al.,2007; Nordgaard et al, 2006; Sundelin et al., 1998; Strauss et al.,2005). There is further evidence to support that, while not the initialtrigger, such defects participate in the pathogenesis of other retinaldegenerative diseases like Stargardts, Bests, and Lebers Amaurosis(Boulton, et al., 2007).

Restoring or augmenting any of these processes may ameliorate or preventthe further deterioration of the retina by minimizing the progression ofphotoreceptor degeneration. Doing so would ultimately protectindividuals from the possibility of further blindness.

In this study, the ability of umbilicus-derived cells to augmentphagocytosis was assessed in an in vitro model that mimics properties ofthe back of the eye.

Materials and Methods

Cells: Frozen vials of previously expanded human adult RPE (ARPE-19,American Type Culture Collection, Manassas, Va.) or primary human RPEisolated from donor cadaver eyes (National Disease Resource Interchange,Philadelphia, Pa.) were plated onto 24 well tissue culture treatedplastic plates in Growth Medium at 20,000 cells/well.

Frozen vials of previously expanded umbilicus-derived cells (passage7-10) were thawed, washed with PBS, and plated into transwells at 10,000or 30,000/well in Growth Medium (0.4 μm pore size PET track-etchedmembrane cell culture inserts, BD Falcon). The transwells were thenadded above the existing RPE cultures in the multiwell plate. Inaddition, previously expanded human dermal fibroblasts (passage 7-10,Cambrex, Walkersville, Md.) were used as a comparison toumbilicus-derived cells.

Co-cultures were maintained at 37 deg C., 5% CO₂ in incubators for 3days to allow for growth factors to be transferred across the membranebetween postpartum cells derived from umbilical tissue and RPE cells.After that time, (1) transwells were either removed and the phagocytosisassay performed, or (2) transwells were removed and well containing RPEalone were maintain for 1 or 4 more days by themselves prior toperforming the phagocytosis assay.

Phagocytosis Assay: This assay was performed utilizing a commerciallyavailable kit (Vybrant Phagocytosis Assay, V-6694, Invitrogen), designedto provide a model system for quantitating the effects of drugs or otherenvironmental factors on phagocytic function. Following removal oftranswells, left over media in bottom of wells was removed and 200 μl ofE. coli reagent was added to each well. This solution containedfluorescently tagged, heat inactivated E. coli prepared as described inthe kit directions. The plate was placed back in the 37° C. incubatorfor 2-3 hours. Following incubation, plates were removed, E. coliparticle solution removed from each well, and trypan blue (200 μl) addedto each well for 1 minute at room temperature to quench any undigestedfluorescent E. coli . Next, trypan blue was removed and wells werewashed with PBS one time. Fresh PBS was added and ingested E. coli wasquantitated by measuring fluorescent activity utilizing a plate reader(SpectraMax® M5, Molecular Devices, limits of excitation 480 nm,emission 520 nm, cutoff 515 nm). Plate reader results were normalized tomedia only controls and quantitatively represented as percentage of thecontrol cell condition, RPE alone, where 100% of control would representequal levels of phagocytosis between compared conditions.

Results

An analysis of phagocytosis was performed assessing the ability ofumbilicus-derived cells to effect levels of phagocytosis in cadavericprimary human RPE (age 76 donor) or a human cell line (ARPE-19, ATCC).Both low passage (passage 2) primary human RPE and ARPE-19 behavedsimilarly when co-cultured with umbilicus-derived cells in thistranswell format assay (for a schematic view, see FIG. 1).Umbilicus-derived cells stimulated phagocytosis versus RPE alone in adose dependent manner after three days co-culture (10,000umbilicus-derived cells=approx doubling in amount of phagocytosedparticles; 30,000 umbilicus-derived cells greater than 2.5 timesincrease in phagocytosis versus control) (FIG. 2). These values werestatistically significant over control, hRPE alone (*p<0.05). However,when umbilicus-derived cells were removed after three days, and the RPEcultures maintained for another 4 days (7 day assay total), overalllevels of phagocytosis went down to control levels (FIG. 2).

In addition, we assessed whether other cell types could stimulatephagocytosis similar to umbilicus-derived cells. In particular weexamined expanded human dermal fibroblasts (Cambrex Biosciences,Walkersville, Md.). In contrast to umbilicus-derived cells treatment, 3day treatment with human dermal fibroblasts led to no changes inphagocytosis above control, RPE alone (FIG. 3, * p<0.05).

Summary: A dose dependent, significant increase in phagocytosis wasobserved after three days co-culture of umbilicus-derived cells withaged (76 year old donor) primary human RPE or an expanded human cellline ARPE-19. The transwell assay performed here suggests that thiseffect was mediated through trophic means rather than cell contact. Thisresult was lost after removal of umbilicus-derived cells for a period of4 days further implicating trophic factors in the mechanism of actionsince most growth factors are labile and would not be expected to havean impact after a further four days in culture. Finally, the observedeffect of umbilicus-derived cells on RPE phagocytosis could not bereplicated using human dermal fibroblasts suggesting that the effect wascell type specific. These results suggest that umbilicus-derived cellssecrete growth factors that stimulate phagocytosis in human retinalpigmented epithelial (RPE) cells.

EXAMPLE 23 Evaluation of the Ability of Umbilicus-Derived Cells toRescue Phagocytic Function In Vitro using Cells from the RCS Loss ofFunction Mutant

The details of the RCS model of retinal degeneration are well describedearlier in this patent application. The merTK defect associated withthis model renders these rats incompetent to phagocytose the shed outersegments of photoreceptors. Previously in Example 22 we have shown theability of umbilicus-derived cells to stimulate human RPE phagocytosisin aged, but otherwise healthy RPE or similar cell lines. Here we testedwhether we could rescue this function utilizing RPE cells from the RCSrodent, a model in which the RPE have a mutation that severely limitsthis phagocytic function and has a known orthologue in humans thatcauses retinitis pigmentosa (Gal et al., 2000).

Materials and Methods

Cells: Primary rat RPE from RCS-dystrophic rats (post natal day 11) wereisolated and plated as previously described (McLaren et al. IOVS1993:34; 317-326; McLaren IOVS 1996:37; 1213-1224). They were culturedfor one week until confluent prior to co-culture with umbilicus-derivedcells. RPE from congenic, healthy rodents were harvested and utilized asa control.

Phagocytosis Assay: Previously expanded and frozen umbilicus-derivedcells (passage 7-10) were next plated on top of RCS—RPE in a similartranswell format as in Example 22. Co-cultures (in the absence ofcontact between the two cell types) were maintained for 24 hours at 37deg C., 5% CO₂. FITC-labeled photoreceptor outer segments (POS) werenext applied to cultures for 3-6 hours at 37 deg C. Cultures were thenanalyzed under an epifluorescent microscope for ingestion of FITC-POS at12.3 hours total assay time post application of FITC-POS.

Quantitation: Quantitative analysis of ingested FITC-POS was performedmanually as previously described (McLaren et al. IOVS 1993:34; 317-326;McLaren IOVS 1996:37; 1213-1224). Briefly, random fields of type I RPEwere taken and the number of ingested particles counted. Controlcongenic (n=6) conditions were compared to dystrophic untreated (n=5)and dystrophic-umbilicus-derived cells treated (n=5) at 40× power. Groupmeans were analyzed for differences using a student's t-test (*p<0.05).Results shown were normalized to congenic controls (100%) for visualcomparison.

Results

A quantitative analysis photoreceptor outer segment phagocytosis wasperformed to evaluate the ability of umbilicus-derived cells tostimulate phagocytosis in otherwise defective RPE from the RCS rat.Normalized to congenic control levels (normal), dystrophic untreated RPEhad a drastic decrease in ability to phagocytose the FITC-POS furtherconfirming the mutation leading to loss of function in the RCS model(25.7% of control) (FIG. 4). However, after 24 hours co-culture withumbilicus-derived cells, phagocytosis was restored to normal congeniclevels in dystrophic RPE (119.2% of control, p=0.0015 vs. dystrophicuntreated).

Summary: In this study, the ability of umbilicus-derived cells tostimulate RPE phagocytosis was assessed. Uniquely, this model allowed usto evaluate the effect of umbilicus-derived cells trophic factors on RPEwith a defect in a phagocytic gene. Further, this model allowed us touse the physiologic photoreceptor outer segments to evaluatephagocytosis. These results suggest that trophic factors secreted byumbilicus-derived cells rescue the loss of function inherent in the RPEcells in the RCS model of retinal degeneration. These results furtherimplicate umbilicus-derived cells in treating a variety of retinaldegenerations associated with phagocytic defects in RPE (Gal et al.,2000; Inana et al., 2004; Inana et al., 2007).

EXAMPLE 24 Attachment of Umbilicus-Derived Cells to Aged Bruch'sMembrane: Ability to Repopulate Degenerate Retina and Sustain RPE CellSurvival or Act like RPE in the Prevention of AMD

Aging changes occur more prominently in submacular Bruch's membrane.Thus, because AMD related changes predominate in the macular region wesought to examine the ability of umbilicus-derived cells to survive andgrow on submacular Bruch's membrane. A demonstration ofumbilicus-derived cells to attach to submacular membranes would provideevidence that these cells could survive in this environment in vivo. Theattachment of umbilicus-derived cells would provide a potential tointegrate and function like RPE or alternately provide a matrix platformon which RPE could repopulate and function.

Previous studies utilizing a Bruch's membrane explant model system showthat retinal pigment epithelial cells have limited capacity to surviveon aged Bruch's membrane, even when a robust fetal cell line is used.These results predict that RPE transplant in patients, particularlypatients with age-related macular degeneration (AMD), will not beeffective. In fact, combined RPE transplantation and choridalneovascular membrane excision has been attempted in AMD eyes, but it hasnot led to significant visual improvement in most patients. In contrast,RPE transplantation in animal models of retinal degeneration has beenproved to rescue photoreceptors and preserve visual acuity. Althoughanimal studies validate cell transplantation as a means of achievingphotoreceptor rescue, laboratory animals in which RPE transplantationhas been successful do not accurately reproduce the age-relatedmodifications of Bruch's membrane in human eyes, which may have asignificant effect on cell graft survival.

With normal aging, human Bruch's membrane, especially in the submacularregion, undergoes numerous changes (e.g., increased thickness,deposition of extracellular matrix and lipids, cross-linking of protein,non-enzymatic formation of advanced glycation end products). BMthickness appears to increase linearly with aging. Membranous debris,filamentous material, and coated vesicles accumulate primarily in theinner collagenous layer by early adulthood and continue to do sothroughout adult life and by late middle age, lipid deposition in BM isapparent. Basal laminar deposit, which comprises mostly wide-spacedcollagen and other materials including laminin, membrane-bound vesicles,and fibronectin, is present in the 7th decade during normal aging. Lipidaccumulation in BM begins to increase significantly after age 40 years.

The results of RPE cell attachment and survival on aged Bruch's membraneand similar preliminary data using putative RPE derived from embryonicstem (RPE-ES) cells indicate poor survival may not be unique to RPE.Additionally, morphology of RPE-ES indicates that some of the cells maybe dedifferentiating or trans differentiating following seeding ontoBruch's membrane. These results indicate the importance of studying thebehavior of postpartum cells derived from umbilical tissue on Bruch'smembrane to determine applicability to treating patients with AMD.

Materials and Methods

The external surface of donor human eyes was trimmed to remove muscle,connective tissue and fat, and the globes were immersed in 10% povidoneiodine briefly. This was followed by washing in BSS. This was followedby two 10-minute incubations in Dulbecco's modification of Eagle'sMedium (DMEM) containing 2.5 mg/ml amphotericin B. The anterior segment,vitreous, and the retina are dissected out. Posterior segments weretrimmed to include submacular Bruch's membrane.

The RPE were gently removed from Bruch's membrane without damaging theRPE basement membrane. After dissecting out the anterior segment,vitreous, and retina from donor eyes, submacular RPE was debrided gentlyusing a microsurgical sponge (Alcon, Fort Worth, Tex.) to create asurface with intact native RPE basement membrane.

Umbilicus-derived cells were seeded at a density of 3146 cells/mm² ontothe submacular explants. Supplemented DMEM was changed every other dayfor 1, 2, 7 or 14 days. After culture of postpartum cells derived fromumbilical tissue on submacular explants, they were fixed in 2%paraformaldehyde and 2.5% glutaraldehyde. Explants were bisectedfollowing minimum overnight fixation and then examined by lightmicroscopy or scanning electron microscopy (SEM). SEM image acquisitionwas performed on a JEOL 35C equipped with a digital image acquisitionsystem (Gatan Inc., Pleasanton, Calif.). SEM analysis focused oncomparing surface morphology of the two cells, confirming the presenceof RPE basement membrane, and determining the extent of cell coverage.

Results

Umbilicus-derived cells survived and grew to confluence on explants forup to 14 days in vitro (FIG. 5). These results demonstrated thatumbilicus-derived cells can repopulate aged Bruch's membrane and thus,providing a potential platform to support RPE repopulation oralternately a replacement for lost RPE.

Summary: Umbilicus-derived cells can repopulate aged Bruch's membraneand thus, provide a potential platform to support RPE repopulation oralternately a replacement for lost RPE.

EXAMPLE 25 Comparison of Integrin Expression Profiles ofUmbilicus-Derived Cells with RPE

Umbilicus-derived cells and ARPE-19 were analyzed for the expression ofseveral integrins to determine if umbilicus-derived cells share similarcharacteristics to RPE.

Materials and Methods

Single cell suspensions of cultured human umbilicus-derived cells andadult retinal pigment epithelial (ARPE-19) cells were prepared bydetaching cells from culture dishes with trypsin 0.5% - EDTA solution(Invitrogen, Carlsbad, CA). Cells were incubated with FACSFlow™ Buffer(Becton Dickinson, Franklin Lakes, NJ) containing 3% fetal calf serum(blocking solution) for 30 minutes at 4° C. with one of the followingmouse monoclonal antibodies (Table 25-1): α1, α2, α3, α4, α5, αvβ3,α2β1, β1 and α5β1. All washes and incubations were done in blockingsolution at 4° C. Non-conjugated primary antibody labeled cells werewashed and incubated in PE-conjugated goat anti-mouse secondary antibodyf( )r 15minutes. Isotype controls contained cells incubated inPE-conjugated goat anti-mouse secondary antibodies (Table 25-2). Atleast 10,000 cells were analyzed with a Becton Dickinson FACSCalibur™flow cytometer (Becton Dickinson, Franklin Lakes, N.J.).

Results

The results demonstrated that umbilicus-derived cells express severalintegrins associated with both fetal and adult RPE (Table 25-1). Theseresults clearly demonstrate that umbilicus-derived cells express thenatural matrix molecules that would allow them to integrate and adhereto Bruch's membrane as was demonstrated in Example 24. Furthermore, theintegrin expression is similar to that previously published for fetalRPE.

Summary: These data suggest that umbilicus-derived cells administrationto the eye may enhance the grafting of an adult RPE celltransplantation. Furthermore, the results confirm that umbilicus-derivedcells express the relative cell surface markers necessary for them toadhere to Bruch's membrane.

EXAMPLE 26 Effect of Umbilicus-Derived Cell Treatment on Phagocytosis inAged Human RPE—Gene Expression Analysis

Age-related macular degeneration (AMD) affects approximately fifteenmillion people over the age of sixty and two million new cases arediagnosed each year. Those stricken by this disease experience adecrease in visual function, leading to blindness.

In this condition, both retinal pigment epithelium cells (RPE) andphotoreceptors are affected. The interaction of the RPE andphotoreceptor outer segments is crucial for the function and survival ofthe photoreceptors. Normally, RPE function to synthesize and secretecomplement factor H(CFH) as well as to ingest and degrade shedphotoreceptor outer segments. Failure of RPE in phagocytosis leads tophotoreceptor cell death, as observed in the Royal College of Surgeons(RCS) rat (Dowling and Sidman, 1962). In the RCS model, a mutation inthe gene Mertk results in the normal binding of photoreceptor outersegments but the inability to ingest shed tips of photoreceptor outersegments. As oxidized shed segments build up, synthesis and secretion ofCFH by RPE is affected. The role of CFH is to regulate complementactivation. If inhibited, CFH-mediated protection of RPE cells may bereduced thus leading to the onset of AMD (Chen, 2007).

In order to assess the impact umbilicus-derived cell application has onhuman aged RPE, differences in host gene expression were examined Humanaged RPE were used in this study for the following reasons: [1] AMDgenerally affects people over the age of sixty and [2] evidence suggeststhat with age, lipofuscin build-up occurs. Lipofuscin build-up in thelysosomal compartment of RPE may cause insufficient synthesis of CFHthus resulting a lack of protection against complement damage.Therefore, a panel of genes associated with phagocytosis, proteosomaldegradation and inflammation was established and subsequent geneexpression levels were compared.

Materials and Methods

Human retinal pigmented epithelium (hRPE) isolation: The hRPE wasdissected from one set of human eyes (75 year old Caucasian female). Thedonor had no known diagnosed eye disease. However, after examining thegross appearance of the retina and RPE, small retinal hemorrhages werenoted (hallmark characteristic of those affected by AMD). The eyes wereobtained through the National Disease Research Interchange (NDR1). Afterremoving the sclera from both eyes, the hRPE was dissected away from theretina. The hRPE was cut into pieces and enzymatically digested for 30minutes in a solution containing trypsin (JRH Bioscience), kynurenicacid ([20 mg], Sigma) and vitrase ([200 units], ISTA Pharmaceuticals).To further disassociate the tissue, cells were triturated and thenbriefly vortexed. To stop the digestion process, DMEM-low glucose media(Invitrogen, Carlsbad, Calif.) containing 15 percent (v/v) fetal bovineserum (FBS; Hyclone, Logan, Utah) was added. After that cells wereforced through a 100 μm nylon cell strainer (BD Falcon). The resultingsolution was then centrifuged for 5 minutes at 250×g. The supernatantwas removed and the cells were resuspended in complete medium containingDMEM-low glucose (Invitrogen, Carlsbad, Calif.), 15 percent (v/v) fetalbovine serum (FBS; Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol(Sigma, St. Louis, Mo.), and penicillin/streptomycin (5,000 Units/mL).Cells were counted with a hemocytometer and plated (concentration of1×105 cells/well) into a six-well laminin coated plate (BD Biosciences).The plate was then placed into a 37° C. incubator.

Preparation of Umbilicus-Derived Cells and Co-Culture Assay: FrozenAliquots of previously expanded umbilicus-derived cells (passage 7) werethawed, washed, counted and seeded (5×10⁴ cells/inserts) into either a6-well cell culture plate (Corning) or into cell culture inserts (poresize: 0.4 μm, BD Biosciences, Franklin Lakes, N.J.). All cells weregrown in the same complete medium as hRPE. The umbilicus-derived cellsinserts were placed directly on top of three wells containing hRPE. Theplates were then placed into a 37° C. incubator for 3 days.

RNA Isolation: After 3 days, the inserts were removed and all cells(hRPE and umbilicus-derived cells) were trypsinized, centrifuged,resuspended and counted. The untreated hRPE and treated hRPE were lysedby addition of 350 μL RLT Buffer (RNeasy® Mini kit, QIAGEN). The lysatewas applied onto a QlAshredder spin column and placed in a 2mLcollection tube and then centrifuged for 2 minutes at maximum speed(18,000g; Microfuge 18 Centrifuge, Beckman-Coulter Cat# 367160). Onevolume of 70% ethanol (200 proof, Sigma, Cat # E7023-500ML) was added tothe homogenized lysate and applied to an RNeasy® mini column placed in a2mL collection tube (supplied). The column was centrifuged for 15seconds at greater than or equal to 8000×g (greater than or equal to10,000 rpm). The flow through was discarded. Buffer RWI (700 μL) wasadded to the RNeasy® column and centrifuged for 15 seconds at greaterthan or equal to 8000×g (greater than or equal to 10,000 rpm) to washthe column. The flow through was discarded. The RNeasy® column wastransferred into a new 2mL collection tube (supplied) and 500 μL BufferRPE was applied to the RNeasy® column. The column was centrifuged for 15seconds at greater than or equal to 8000×g (greater than or equal to10,000 rpm). The flow through was discarded. Another 500μL of Buffer RPEwas applied to the RNeasy® column and centrifuge for 2 minutes atgreater than or equal to 8000×g (greater than or equal to 10,000 rpm) todry the RNeasy® column. The RNeasy® column was transferred to a new1.8mL collection tube (DNA LoBind tube 1.5 mL 22 43 102-1, Eppendorf AG)and centrifuged in a microcentrifuge at full speed for 1 minute. Toelute the RNA from the RNeasy® column, the column was transferred to anew 1.5mL collection tube (supplied) and 30 μL RNase-free water wasapplied. The tube was then centrifuged for 1 minute at greater than orequal to 8000×g (greater than or equal to 10,000 rpm). Aftercentrifugation, the tube was removed, capped and labeled and thentransferred to a −80° C. freezer.

RNA quality and quantification: The quantity and quality of RNA wasdetermined using SOFTMAX Pro software on the SPECTRAMAX M5spectrophotometer (Molecular Devices). Deionized water (dH20) was usedas a blank and was loaded into the first well of a 96-well UV plate(Costar, Corning). Diluted RNA samples (1:20) were loaded intosubsequent wells and the reading was taken. The RNA concentration wascalculated: (μg/mL)=(OD260)×(dilution factor)×(40 μg RNA/ml). Then 10 μLof each diluted RNA sample was loaded into an agarose E-gel (Invitrogen)along with 10 μL of TrackIt (50 bp DNA ladder, Invitrogen). Samples wererun for 15 minutes. After time had expired, images were taken of the gelusing the Chemi Doc XRS camera system (Bio-Rad).

First strand cDNA synthesis: The synthesis of cDNA was performed usingthe SUPERSCRIPT III First-Strand Synthesis System (Invitrogen, Carlsbad,Calif., USA). Reagents were combined and added to each tube as shown inTable 26-1.

Tubes were then transferred to a Thermal Cycler (Bio-Rad Cat#170-9703),incubated at 65° C. for 5 minutes and then cooled to 4° C. Next, 20 μLof cDNA Synthesis Mix (Table 26-2) was added to each tube. The tubeswere then heated to 50° C. for 50 minutes, followed by another 85° C.cycle, which lasted for 5 minutes, and finally a cooling 4° C. cycle for15 minutes. At this time, 1 μL of RNase H was added to each tube. Thetubes were again incubated for 20 minutes at 37° C. At the end of thiscycle, the cDNA synthesis reaction was used immediately for RT-PCR.

Real-time quantitative RT-PCR: The mRNA levels of the analyzed geneswere quantified by real time RT-PCR. Master mix was made (Table 26-3)and then pipetted into a Fast Thermal Cycler Optical 96-well plate(Applied Biosystems).

The RT-PCR reaction was performed using the 7900HT Fast Real-Time PCRSystem (Applied Biosystems). Thermal cycle conditions were as follows:50° C. for 2 minutes, 95° C. for 10 minutes followed by 40 cycles of 95°C. for 15 seconds and 60° C. for 1 minute. At the completion of thereaction, the data was saved and then analyzed with the SDS 2.2.2 systemsoftware (Applied Biosystems).

Results

Genes used for analysis: The names of the genes that were chosen forthis analysis, their abbreviation, catalogue number and proposedfunction in the eye are summarized in Table 26-4. The genes were chosenbased on their importance in functions for RPE.

Real time RT-PCR: For the calibration of mRNA expression levels, GAPDHwas selected to serve as the endogenous control. Thus for analysis, theexpression levels of all genes were normalized to GAPDH.

In this analysis, the gene expression levels of untreated RPE werecompared to umbilicus-derived cell treated RPE (Table 26-5). In hRPEco-cultured with umbilicus-derived cells, increases expression in thefollowing genes were observed: MERTK, CRALBP, INTAV, CATHD and CFH.(2.8±0.2 co-culture vs. 8.2±0.2 control, *p<0.05). These increases wereseen only after 3 days of co-culture with umbilicus-derived cells. Theseresults suggest that umbilicus-derived cell treatment had an impact onthe expression of genes associated with phagocytosis, proteosomaldegradation and inflammation.

Summary: Current physical treatments fail to improve visual function forthose affected by AMD. Retinal repair may require the transplantation ofhealthy cells for the treatment of this disease. Co-culture ofumbilicus-derived cells on aged hRPE has shown to impact the expressionof genes associated with phagocytosis, proteosomal degradation andinflammation.

Accumulating evidence suggests that immunological factors, such as CFH,may play an important role in the development of macular degenerationdevelopment. It has been shown in vitro that freshly plated human RPEcells express high levels of CFH. However, when human RPE cells areexposed to prolonged incubation with oxidized photoreceptors rod outersegments or with pro-inflammatory cytokines such as, TNF-alpha and IL-6,CFH is down regulated (M. Chen et al, 2006). This study suggests thatthe co-culture of umbilicus-derived cells could be used for treatment ofmacular degeneration as well as other ocular indications wherephagocytosis or inflammation may be affected.

EXAMPLE 27 Gene Profiling Approach to Identify Potential Mechanisms bywhich Umbilicus-Derived Cells Preserve Vision in the Royal College ofSurgeons Rat Model of Retinal Degeneration Materials and Methods

Cell culture for trophic factor secretion: Umbilicus-derived cells(population doubling 20) was plated at a seeding density of 10,000/cm²into gelatin-coated 6-well plates in Growth Media as described in theprevious example. After 24 hrs, the culture media was replaced with 1 mlfresh growth media. The supernatant of the culture was collected at day1, 3, 7 and saved frozen at −80° C. Cell number counts were performedfor each individual sample. These culture media were sent to PierceBiotechnology Inc. (Worcester, Mass.) for the analysis described below.

ELISA: ELISA for HGF and IGF followed the instruction from R&D systems.Briefly, working standards were prepared as directed in the instructionmanual (HGF: 125 pg/ml-4000 pg/ml; IGF: 94 pg/ml-6000 pg/ml). Standards,control, or sample (50 μl) were added to each well and were incubatedfor 2 hours at room temperature for HGF and at 4° C. for IGF. The platewas washed four times. 200 μl, of HGF or IGF conjugate was added to eachwell and incubated for 1.75 hours at room temperature for HGF and for 1hour at 4° C. for IGF. The plates were washed for 4 times, then 200 μLof Substrate Solution was added to each well and incubated for 30minutes at room temperature for HGF and IGF. 50 μL of stop solution wasadded to each well. The optical density of each well was determinedusing a microplate reader set to 450 μm.

Cell transplants. The transplantation procedure has been describedabove. Briefly, 20,000 umbilicus-derived cells in 2 μl PBS were injectedinto the dorso-temporal subretinal space of anesthetized 3-week olddystrophic-pigmented RCS rats. As controls, sets of RCS animalsunderwent either a sham injection procedure containing PBS only, or wereleft untreated.

After cell injections were performed, animals were injected withdexamethasone (2 mg/kg) for 10 days post transplantation. For theduration of the study, animals were maintained on oral cyclosporine A(210 mg/L of drinking water; resulting blood concentration: 250-300micrograms/L) (Bedford Labs, Bedford, Ohio) from 2 dayspre-transplantation until end of the study. Food and water wereavailable ad libitum. Animals were sacrificed at day 7, 30 and 60 posttransplantation (dystrophic-untreated, dystrophic-cell injected) formicroarray study. Sham-operated animals were only collected at day 7post injection

RNA sample collection. Animals were sacrificed at day 7, day 30- and day60-post cell transplantation, with an overdose of urethane (12.5 g/kg).Eyes were extracted from the animals and fixed in RNALater to preventRNA degradation. RNA extraction was performed following InvitrogenTrizol protocol. RNA concentration and quality were examined by BIO-RADExperion Bioanalyzer. From each sample, 2 μg total RNA was used forAffimetrix rat 230 version 2 chips. Three sets of comparisons were done:day 7 treated vs. sham; day 30 treated vs. non-treated; day 60 treatedvs. non-treated. In each group, three individual samples were used.

Microarray analysis: The RNA samples were sent to Johnson & JohnsonPharmaceutical Research and Development in La Jolla, Calif. where theAffymetrix rat chip was used for microarray analysis. The raw microarraydata was normalized across the 16 chips using Quantile-Quantilenormalization. ANOVA and Multiple test correction (FDR; p-value <=0.01),two methods provided in the Partek Pro (St. Louis, Mo.) software, wereused to determine differences between treatment and time factors afternormalization. Differentially expressed genes were identified for eachtime point using the t-test method (p-value less than or equal to 0.05)in conjunction with fold changes (greater than or equal to 1.5) betweenthe mean values of each group. With differentially expressed genesdetermined, relevant pathway analysis was constructed using Ingenuitysystems (IPA) from Ingenuity Systems (Redwood City, Calif.).

Results

Gene profiling revealed protected photoreceptor function: In order tounderstand the possible mechanisms resulting in the preserved visualfunction in the umbilicus-derived cell treated eyes, we conducted amicroarray study to identify the differentially expressed genes and thecanonical pathways in the treated versus non-treated rat eyes to guidefurther in vitro studies. This study was designed to recognizedifferential rat gene expression and pathway networks in response toumbilicus-derived cell treatment in the eyes. Total RNA was obtainedfrom treated and non-treated rat eyes at different time points (Days 7,30 and 60 post cell transplantation, n=3). At each time point, thenon-treated fellow eyes were used as controls. Eyes from sham-operatedrats were collected at day 7 and were used as controls for Day 7 timepoint. Standard hybridization procedures were performed for AffimetrixRat 230 chip. 23,000 gene expression data were obtained from theAffimetrix Rat 230 chip. Raw data were normalized across all chips. 987genes were differentially expressed in treated eyes vs. controls withstatistic significance (p<0.05). Hierarchical clustering was performedand the heat map was generated. The heat map demonstrated thatindividual sample at the same time point tends to cluster together,suggesting an evident time effect for the treatment and at the same timepoint, treatment groups differ from the control groups.

Based on the analysis described above, different predominating genenetworks were identified at different time points after celltransplantation. At day 7, in the analysis of umbilicus-derivedcell-treated vs. sham-operated eyes, genes involved in cellproliferation and growth such as FOS, CSNK1D, and RPS6KA2 were upregulated in cell-treated eyes, as shown in Table 27-1. FOS is atranscription factor that regulates cell proliferation in many celltypes. Increased FOS expression has been documented in RPE cells duringproliferation. FOS activation is regulated by MAP kinase cascade, whichlinks the growth factor receptor activation at cell surface to thetranscription factor such as FOS in the nucleus. RPS6KA2, also known aspp 90^(rsk), is a kinase involved in the activation of FOS in the MAPKcascade.

The increased expression of a kinase cascade component-pp 90^(rsk) and adown-stream transcription factor—FOS strongly suggests the impact ofumbilicus-derived cells on cell proliferation in the RCS rat eyes. RPEcell proliferation potentially can increase RPE cell number in theretina and potentially enhance phagocytosis, thus, protectphotoreceptors from apoptosis, and preserve visual function.

More over, genes involved in insulin-like growth factor (IGF) pathway,such as IGFBP5, IGF2BP3, were also up-regulated as shown in Table 27-1.A wide range of biological processes are modulated by IGF-1 signalingpathway, including, for example, cell proliferation, tissue-specificdifferentiation, and protection against apoptosis. In RPE cells,activation of IGF cascade is related with reduction of apoptosis-inducedby hydrogen peroxide IGF pathway members such as IGF1 binding protein(IGFBP)₅ and IGFBP3 are well documented to inhibit apoptosis in multiplecell types, including RPE cells. IGFBP5 and IGFBP3 promote theactivation of IGF1 receptor by carrying IGF1 in the circulation andfacilitating binding between IGF1 and its receptor. The up regulation ofIGFBP5 and IGF2BP3 in the treated rat eyes demonstrated that activationof IGF pathway is one of the protection mechanisms induced byumbilicus-derived cells against cell death.

Another molecule that umbilicus-derived cells impacted was TXNL1. TXNL1is thioredoxin-related protein, a member of the thioredoxin pathway,which regulates oxygen stress and protects cells against oxygenstress-induced cell death. Oxygen stress stimulation such as hydrogenperoxide treatment induced apoptosis in cultured RPE cells. Thus, theincreased activity of thioredoxin pathway potentially could release theoxygen stress and protect RPE. Indeed, this pathway has been shown upregulated in non-apoptotic RPE cell line compared to apoptosis RPE. Atday 7-post cell transplantation, the up-regulation of cell proliferationand protection against cell death, either by increasing the activity ofIGF signaling pathway or by releasing oxygen stress, paves the way forpreserved photoreceptors in the outer nuclear layer and preserved visualfunction in RCS rats.

The predominant anti-apoptosis pathways at day 7 continued to be thedominant signaling network at day 30 as shown in Table 27-2. Theplatelet-derived growth factor C (PDGFC) is unregulated at day 30 in thecell-treated eyes. PDGFC is a member of PDGF family. Similar to IGF1,PDGF family members also demonstrate a broad biological functions inmany cell types, including proliferation and survival.

Down-stream to the PDGF receptor, SH2B was up regulated in the treatedeyes. SH2B is a cytoplasmic adaptor protein that connects the growthfactor receptor to the kinase cascade. The up regulation of both thegrowth factor PDGFC and the adaptor protein SH2B demonstrated the impactby umbilicus-derived cells on this growth factor pathway. Thecontinuation of the growth factor activation by the initial treatmentdemonstrated the long-lasting trophic effect from umbilicus-derivedcells in the treated eyes and correlated with the retention ofumbilicus-derived cells in the eyes.

Pathway analysis from Day 60 data revealed a predominantphototransduction pathway in the treated eyes (Table 27-3). Thephototransduction pathway is shown in FIG. 6. Briefly, light activationcauses a graded change in cell surface membrane potential. Thetransmission of the pulses is mediated by opening or closing of ionchannels, which are regulated by cyclic guanosine monophosphate (cGMP).The series of biochemical changes that ultimately leads to a reductionin cGMP levels begins when a photon is absorbed by the photo pigment inthe receptor disks, which contain proteins called opsins. One of opsinsin rod cells that mediate the molecular events post light perception isrhodopsin (RHO). When the retinal moiety in the rhodopsin moleculeabsorbs a photon, its configuration changes. This change then triggers aseries of alterations in the protein component of the molecule, andleads to the activation of an intracellular messenger called transducin,which activates a phosphodiesterase that reduces the concentration cGMPand leads to channel closure at cell surface membrane (FIG. 6).

Molecules involved in the phototransduction pathway such as CNGA1,GNAT1, GNB1, RHO, and PDE6B were up regulated. These genes distributedin the entire photoreceptor transduction network. Cyclicnucleotide-gated (CNG) ion channel subunit CNGA1 expression is increasedmore than 6 fold in the treated eyes and located at photoreceptor outersegment in the transduction pathway to eventually mediate the membranepotential pulses in photoreceptors as shown in FIG. 6. RHO is one of theproteins in opsins, which is a complex of molecules to receive andprocess photons from the light. The up-regulation of RHO suggestspreserved light perception function in rod cells. GNAT1 and GNB1 arealpha and beta units in transducin that couple RHO andcGMP-phoshodiesterase during visual impulses. The expression of othervisual function regulatory molecules, PDE6B and ROCK1, which directlyregulate cGMP were also up regulated. Interestingly, the predominatingpathway transition from anti-apoptotic pathway at day 7 and 30 tophototransduction pathway at day 60, suggests a direct impact ofumbilicus-derived cell treatment on the protection of photoreceptors.

Trophic factors secreted by umbilicus-derived cells: From the microarrayresults, it is evident that umbilicus-derived cells may play a role inpromoting proliferation and reducing apoptosis at early stage.Additionally, umbilicus-derived cells may play a role in preventingphotoreceptor cell death, and maintaining phototransduction at laterstage. To determine the cytokines secreted by umbilicus-derived cellsthat can impact on proliferation and protection, we looked into thetrophic factors that are secreted by umbilicus-derived cells in vitro.

From the growth factor analysis, IL-8, IL-6, HGF, and IGF-1 are highlysecreted by umbilicus-derived cells in vitro (Table 27-4 and Table27-5). The pathway analysis described above has demonstrated theinvolvement of IGF pathway in the umbilicus-derived cell treated eyes.Other cytokines that are secreted by umbilicus-derived cells in vitroinclude basic FGF, BDNF, CNTF, NT3 as shown in Table 27-4. The trophicfactor profiles correlated with the findings from microarray.

EXAMPLE 28 Conditioned Medium from Umbilicus-Derived Cells ProtectedARPE-19 From H₂O₂-Induced Apoptosis

Apoptosis may be involved in the development of retinal degenerationdiseases such as RP and AMD. Apoptosis in photoreceptors is derived frommultiple pathophysiologies, including defects in the process ofphagocytosis and oxygen stress. Studies from animal models have shown adeleterious effect in photoreceptors and RPE from oxidative damages. Theoxidative damage can form a cycle of cell death initiated from the rodundergoing apoptosis. The apoptotic rod cells created environment withincreased blood flow and higher oxygen in the retina, which lead todeterioration of RPE, rods, and cones, which is the major cell type forvision.

In the previous example, a gene profiling study (Microarray) revealedanti-apoptosis as a key signaling pathway at early stage post celltransplantation and preservation of phototransduction at later stage.Follow-up trophic factor assessment in the umbilicus-derived cellconditioned media has confirmed the microarray results and identifiedseveral cytokines as anti-apoptotic agents based on literatures. In thisstudy, we intend to evaluate if umbilicus-derived cell conditioned mediacan protect RPE cell line from apoptosis induced by hydrogen peroxide(H₂O₂) and to develop a cell-based apoptosis assay.

Materials and Methods

Umbilicus-derived cell culture for conditioned media collection: Frozenvials of previously expanded umbilicus-derived cells (populationdoubling 20) were plated at 5,000 cells/cm² on T75 flasks in GrowthMedium as described above. The flasks were incubated in a 37° C.incubator for 24 hours. The supernatant of the culture was collectedafter 24 hour culture, and saved frozen at −80° C. or applied to ARPEcultures immediately.

ARPE-19 culture: Frozen vials of previously expanded ARPE-19 cells(CRL-2502, American Type Culture Collection, Manassas, Va.) were seededonto 24-well plates in Growth Medium at 40,000 cells/well or in 96-wellplate at 5000/well for 24 hours. Cells were washed with PBS and treatedwith media containing H₂O₂.

H₂O₂ treatment: H₂O₂ was prepared by adding H₂O₂ at differentconcentrations (0, 0.125 mM, 0.25 mM, 0.5 mM, 1.0 mM, 1.2 mM) to thegrowth media (GM) or conditioned media (CM) from umbilicus-derived cellculture. ARPE-19 were cultured in H₂O₂ containing media for 0, 0.5 hr, 1hr, 2 hr, 3 hr, then the H₂O₂ media was removed and cells were used forfurther apoptosis assays.

For Annexin V assay, ARPE-19 cells were treated with er growth media orconditioned media for 24 hours before assessment of apoptosis.

Colometric Apoptosis assay: The apoptosis assay was performed accordingto the instruction in the Cell Death Detection ELISA^(plus) kit fromRoche (Cat # 11 774 425 001). Briefly, the cells still attached to theplate was lysed and incubated with an antibody against histone to pullhistone from the whole lysates. Another antibody, linked with HRP,recognizing the DNA fragments will be used to identify the DNAcomponents within the histone section. The apoptotic complex of histoneand DNA fragments will be recognized by HRP substrates.

Annexin Vapoptosis assay: After a 24 hour incubation period, the culturemedium was removed and the plate was washed with PBS. Trypsin (Gibco)was added to dislodge the cells from the individual wells. The culturemedium from each well and the trypsinized cells from each respectivewell were combined and centrifuged at 250×g for 5 minutes and the pelletwas resuspended in 40 μL cold 1× Nexin buffer. Five microliters AnnexinV-PE and 5 μL Nexin 7-AAD were added to each sample and the mixture wasvortexed and incubated shielded from light at 4° C. for 20 minutes.Following incubation, 450 μL of cold 1× Nexin buffer was added to eachsample and the suspension was vortexed. The stained samples were loadedonto the Guava® Personal Cytometer and were acquired and analyzedaccording to the Guava® Personal Cytometer User's Guide.

Results

Time- and dose-dependent apoptosis induction by H₂O₂ in ARPE-19 cells:H₂O₂ induced apoptosis in a time- and dose-dependent manner. Apoptosismeasured by DNA fragments associated with histones was determined usingELISA. When ARPE-19 cells were treated with a series of concentrationsof H₂O₂ from 0.25 mM to 1.2 mM, DNA fragmentation was at 1-fold ofnegative control (0 mM) at 0.5 mM and was increased to 2.6-fold and 3.3fold at 1 mM and 1.2 mM respectively, shown in FIG. 7.

In a separate experiment, apoptosis was measured by measuring thepercentage of Annexin V(+) cells in the population. One of the earlyevents during apoptosis process is the exposure of a phospholipid-likephosphatidylserine (PS) to the cell surface membrane. Annexin V is amolecule that can bind to PS on the cell surface. Using this feature,apoptotic cells can be detected by staining the cell surface withAnnexin V. The percentage of cells in the population that are either inthe early stages of apoptosis or the later stages of apoptosis wasdetermined by measuring changes in cell membrane permeability to the DNAdye 7-AAD. At the early stages of apoptosis, when the cell membrane isstill intact, 7-AAD is unable to penetrate the cell membrane and will benegative. Therefore early apoptotic cells are typified as being AnnexinV(+) and 7-ADD(−). At later stages of apoptosis, when the cell membraneintegrity fails, and can be penetrated by 7-ADD, the apoptotic cells aredemonstrated as Annexin V(+) and 7-ADD (+).

Early apoptotic cells, as determined by the percentage of cells thatwere Annexin V(+) and 7-AAD(−) increased with the concentration of H₂O₂.At 0.125 mM H₂O₂, the percentage of cells that were Annexin V(+) and7-AAD(−) was 8%. This increased to 18% at 0.25 mM H₂O₂ and reached amaximum at 34% at 0.5 mM H₂O₂. Similarly, total apoptotic cells, asdetermined by measuring the total Annexin V(+) cells in the populationwas 16% at 0.125 mM H₂O₂, that increased to 27% at 0.25 mM H₂O₂. Theerect of H₂O₂ on the total number of apoptotic cells was maximal at 0.5mM H₂O₂ (FIG. 8). When ARPE-19 was treated at 1.2 mM but with differentincubation time, a time course effect was evident. DNA fragmentationreached its plateau by 1 hr, about 3.3-fold versus 0 hr incubation, andremained at this level to 3 hrs as shown in FIG. 9.

Umbilicus-derived cell conditioned media reduced apoptosis both dose-and time-dependently: The DNA fragmentation induced by H₂O₂ wasdecreased when cells were treated with conditioned media fromumbilicus-derived cells. DNA fragment levels at different doses anddifferent incubation time in conditioned media were maintained at thesimilar level as the negative control from 0.25 mM to 1.2 mM (0.5-foldto 1.5-fold vs. control), shown in FIGS. 7 and 9.

Similar results were seen using the Annexin V assay. Early apoptosisevents and total apoptotic cells were reduced in umbilicus-derived cellconditioned media-treated ARPE-19 cells at 0.25 mM, 0.5 mM, and 1.0 mMas shown in FIG. 8.

These results confirmed the findings in literature that H₂O₂ is a potentstimulator of apoptosis for RPE cells. Most importantly, conditionedmedia from umbilicus-derived cells reduced apoptosis measured by bothDNA fragmentation and Annexin V positivity. The dose- and time-dependentinhibition of apoptosis by the conditioned media strongly suggests thatcytokines secreted by umbilicus-derived cells can rescue RPE cells fromoxygen stress-induced apoptosis.

EXAMPLE 29 Preservation of Multiple Cell Types in the Retina of aPreclinical Model of Retinitis Pigmentosa

The Royal College of Surgeons (RCS) rat is a preclinical model ofretinitis pigmentosa in which there is progressive degeneration of therod and cone photoreceptors resulting from a specific defect in theretinal pigmented epithelial cells (RPE). In this model, a mutation inthe allele of the gene for the receptor tyrosine kinase Mertk results inan inability of the RPE to phagocytose shed rod outer segments (ROS).This defect results in apoptotic photoreceptor cell death, beginningaround post-natal day 20 (P20), and by P60 the outer nuclear layer (ONL)of these animals, which contains the photoreceptor cell nuclei, is only1-2 layers in thickness.

Umbilicus-derived cells are an allogeneic cell type with potential forcell therapy applications. Umbilicus-derived cells are obtained from anethical cell source and can be readily expanded to yield at least 1×10¹⁷cells from a single donor without karyotypic or phenotypic changes. Asingle dose of 20,000 umbilicus-derived cells into the subretinal spaceof RCS rats can preserve visual function as assessed by the use ofelectroretinogram, optomoter and luminance threshold testing (See, forexample, Lund et al. 2007). Umbilicus-derived cells have alsodemonstrated superior efficacy in preserving visual function compared totwo other allogeneic expandable tissue-derived cell types;placental-derived cells and human bone marrow mesenchymal stem cells(hMSC) (Lund et al. 2007). Furthermore, anatomical analysis demonstratedthat umbilicus-derived cells at 80 days post cell injection can preserve4-5 layers of cell nuclei in the outer nuclear layer (ONL) of thesubretinal space compared to sham or untreated controls which at thesame time point only have 1 layer of cells retained in the ONL.

While histology has been used to visualize the effects ofumbilicus-derived cell therapy on the retina, it has been mostlyanecdotal in nature and not quantitative. The goal of this study was touse quantitative morphometry to characterize the preservation of variouscell types in the retina important in rod photoreceptor connectivity andsignaling following subretinal umbilicus-derived cell administration inRCS rats.

Materials and Methods

Animals: Experiments were performed on male and female pigmenteddystrophic RCS rats (rdy−/p−), which were individually housed with a12-hour light/dark cycle at the Moran Eye Center (University of Utah,Salt Lake City, Utah). All procedures were approved and monitored by theUniversity of Utah Institutional Animal Care and Use Committee and havebeen conducted in accordance with the Policies on the Use of Animals andHumans in Neuroscience Research, revised and approved by the Society forNeuroscience in January 1995. The rats weighed approximately 30-40 gramsat age of dosing (post-natal day 21). Food and water were available adlibitum.

Preparation of Donor Cells: Human Umbilical Cords were Obtained withDonor consent following live births from the National Disease ResearchInterchange (Philadelphia, Pa.) and umbilicus-derived cells wereisolated, tested and cryopreserved as described in Examples 1-5 above.Prior to injection, umbilicus-derived cells were thawed rapidly in a 37°C. water bath, washed twice in sterile PBS and resuspended at a finalconcentration of 1×10⁴ cells/μL.

Injection procedure: On Day 0 (post-natal Day 21) the rats received asubretinal injection of 20K umbilicus-derived cells in a volume of 2 μL.RCS rats were anesthetized with an intraperitoneal injection ofxylazine-ketamine (1 mg/kg of the following mixture: 2.5 ml xylazine at20 mg/ml, 5 ml ketamine at 100 mg/ml, and 0.5 ml distilled water). 20Kumbilicus-derived cells were injected through a fine glass pipette(internal diameter 75-150 μm) into the eye through a small scleralincision. The incision was closed with a small suture after injectionwas completed. All animals received daily dexamethasone injections (1.6mg/kg, i.p.) for 2 weeks post-injection, and received cyclosporine-A(Bedford Labs, Bedford Mass.) administered in the drinking water (210mg/L; resulting blood concentration: 250-300 μg/L) from 1-2 days priorto cell injection until euthanasia. Rats were sacrificed at thefollowing time points after surgery: 1, 7, 14, 30 and 60 days.Euthanasia was performed by anesthesia with a ketamine:xylazine:acepromazine mixture, followed by exsanguination.

Collection of eyes and extraction of RNA for RT-PCR: Followingeuthanasia, the eyes were extracted and placed in RNAlater solution(Ambion, Austin, Tex.). All eyes were kept at 4° C. in RNAlater solutionfor up to 3 days, then stored at −80° C. until processing. Eyes wereplaced in Lysing Matrix tubes (Qbiogene, Carlsbad, Calif.) containing1.4ml Trizol (Invitrogen, Carlsbad, Calif.) and centrifuged in a Fast PrepFP120 homogenizer (Qbiogene, Carlsbad, Calif.) at speed #6 for 45 sec.The supernatant was transferred to a 5 ml round bottom tube. Trizol (2-4ml) was added to the supernatant and samples were then centrifuged at9000 rpm for 10 min. Chloroform (2-3 ml) was added to the supernatantand incubated at room temperature then centrifuged at 9000 rpm for 10min at room temperature. Isopropanol (3-4 ml) was added to thesupernatant to precipitate total RNA. The RNA pellet was obtained bycentrifuging at 9000 rpm for 10 min. The pellet was washed with 70%ethanol and air-dried at room temperature, then resuspended in 100 μlRNase-free water and stored at −80° C. The RNA quality and quantity wasanalyzed using the Experion automated electrophoresis system (Bio-RadLaboratories, Hercules, Calif.).

2-step RT-PCR: First-strand cDNA synthesis was performed using theFirst-Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Briefly, 10μg total RNA was mixed with 2 μl oligo(dT)20 (50μm) and 2 μl dNTP (10mM), incubated at 65° C. for 5 min, then added 4 μl 10× reaction buffer,8 μl 25 mM MgC12,4 μl 0.1 M DTT, 2 μl RNaseOUT and 2 μl SUPERSCRIPT IIRT enzyme, incubated at 50° C. for 50 min, and 65° C. for 5 min.Template RNA was removed by addition of 1 μl RNase H for 20 min at 37°C. Real-time PCR was performed on the 7900HT Fast Real Time PCR System(Applied Biosystems, Foster City, CA) using human β-2 micro globulin(hβ2M) TaqMan® Gene Expression Assay with the TaqMan® Fast Universal PCRMaster Mix (Applied Biosystems, Foster City, Calif.). All samples wereanalyzed in triplicate. RNA standards were run alongside samples forquantitation of the number of cells present in injected eyes. Togenerate RNA standards, eyes from Sprague-Dawley rats were collected asdescribed above. The eyes were injected with 160, 800, 4K, 10K, and 20Kumbilicus-derived cells (n=3). RNA extraction and two-step real-timeRT-PCR was performed as described above. The threshold cycle value wasobtained and analyzed against the injected cell numbers.

Collection and processing of eyes for histology: Following euthanasia,the rat was flushed with phosphate buffered saline under low pressurevia the aorta. The eyes were carefully removed, and surrounding tissuefrom the eye trimmed. Both the right and the left eye from each animalwere collected and immersed in Pen-Fix fixative (Richard AllanScientific) for 24-48 h. After fixation, the eyes were processed forhistology including dehydration, clearing in xylene and infiltrationwith paraffin. Each eye was specifically oriented during embedding inparaffin: the superior pole of the eye was embedded down in the paraffinblock and the suture marking the injection site rotated in a clockwisemanner until it was approximately in the 3 o′clock position on theglobe. This orientation was maintained during embedding. This resultedin the injection site residing approximately in the central plane of theeye for the majority of the eyes processed. For injected eyes,collection of 5 μm sections began after facing into the block until thesuture marking the injection site is identified on a stereomicroscope(FIG. 10). If no suture was identified, the eye was excluded fromfurther analysis, as was its uninjected counterpart (left eye from sameanimal). If the suture was identified after sectioning through thecentral plane of the eye and it was determined that it resided near thepole of the eye, it was excluded from further analysis, as was itsuninjected counterpart. For uninjected eyes, sectioning began after thearea/depth of interest is identified in the injected (right) eye. Thecorresponding uninjected (left) eye from the same animal was sectionedto a similar depth, collecting sections from approximately the same areaof the eye.

Antibodies: The following antibodies were used for immunohistochemistry:rabbit-anti-Rhodopsin (Chemicon), rabbit anti-Calretinin (Chemicon),rabbit anti-Recoverin (Chemicon), mouse-anti-human nuclear matrixantigen (NuMA, Calbiochem). Antibodies for secondary detection werebiotinylated anti-rabbit (Chemicon), and biotinylated goat-anti-mouse(Jackson Immunoresearch). Immunocytochemistry: Sections for IHC stainingwere incubated for 1 hr at 59° C. followed by deparaffinization througha series of changes in xylene, 100% alcohol, 95% alcohol, and water on aTISSUE TEK® DRS™ 2000 Slide Stainer (Sakura, Torrance, Calif.). Slideswere rinsed with tap water for approximately 5 minutes. Allimmunohistochemical staining was performed on the i6000™ AutomatedStaining System (BioGenex, San Ramon, Calif.). Antigen retrieval wasperformed when necessary using a Decloaking Chamber (BioCare Medical,Concord, Calif.) and Reveal HIER Solution (BioCare Medical) ormicrowaved using Antigen Retrieval Citra Solution (BioGenex). Endogenousactivity of peroxidase and antigenic sites were blocked and normal goatserum (BioGenex) reduced background staining due to non-specific bindingof the primary or secondary antibody. Sections were incubated withprimary antibody at room temperature, detection of the bound primary isachieved by the addition of a biotinylated secondaryantibody+peroxidase-conjugated streptavidin (BioGenex) and peroxidaseactivity was made visible with diaminobenzidine (DAB) (BioGenex).Counterstaining with Mayer's Hematoxylin (BioGenex) for 1 min.

Image acquisition and analysis: A Nikon Eclipse E800 (Nikon Corporation,Tokyo, Japan) microscope was equipped with an Evolution™ MP 5.0 RTVcolor camera (Media Cybernetics, Inc. Silver Spring, Md.), interfacedwith an IBM computer (International Business Machines Corporation,Armonk, N.Y.) running Windows 2000 (Microsoft Corporation, Redmond,Wash.). Images were captured and analyzed using Image-Pro Plus softwareversion 5.1 (Media Cybernetics, Inc. Silver Spring, Md.). MicrosoftExcel 2000 (Microsoft Corporation, Redmond, Wash.) and GraphPad Prismversion 4.03 (GraphPad Software, Inc. San Diego, Calif.) were used tointerpret, analyze and graph the raw data. SigmaStat StatisticalSoftware version 2.03 (SPSS, Inc. Chicago, Ill.) was used to performstatistical analysis on the collected data. Using the Auto-Pro toolwithin the Image-Pro Plus software, custom written macros were used toperform the analysis consistently.

Morphometry of day 60 eyes: Three umbilicus-derived cell injected andthree control (uninjected) eyes from the day 60 group (4 animals) wereimaged and analyzed with morphometry. The images captured were 24-bitRGB images, 2560×1920 pixels in size, with a resolution of 300×300dots/inch. The images were captured with a 60× objective Nikon lens. Noimaging or analysis was performed on areas of the retina that were torn,damaged, folded or missing.

Measurement of area of outer nuclear layer (ONL) per length: From eacheye, three hematoxylin and eosin (H&E) stained sections, each separatedby 20 μm in depth were imaged and analyzed for the ONL measurements. Theareas where the images were collected were defined as regions 1 and 2.Region 1 is an area near the injection site and region 2 is an area awayfrom the injection site. Up to ten images (five from each region) werecollected from each section. Using Image-Pro Plus, the ONL was selectedas the area of interest (AOI). The AOI was extracted and transformedinto an 8-bit grayscale image, referred to as a mask. Area and length(to normalize the data) of the ONL were measured and used to calculatethe area per length of the ONL for each image.

Measurement of Amount of rhodopsin immunostaining in the neuroepitheliallayer: One section per eye immunohistochemically stained for rhodopsinwas imaged and analyzed. The area where the images were collected wasdefined as region 1, an area near the injection site. Up to eight imageswere collected from each section. Using Image-Pro Plus, the saturationchannel of the color model HSI was extracted from the RGB image. Usingvarious acquired images with mixed saturation levels, a threshold wasset to the most intensely stained colors or the most dominant hues. Thisset threshold was used to analyze each image. The AOI was defined as theneuroepithelial layer in each image. The measurement calculated for eachimage was the percent area of the AOI that was within the set threshold.

Measurement of Area of the calretinin immunostaining per length: Onesection per eye, immunohistochemically stained for calretinin was imagedand analyzed. The area where the images were collected was defined asregion 1, an area near the injection site. Up to eight images werecollected from each section. Using Image-Pro Plus, three AOIs weredefined as: all recoverin-positive areas, the inner nuclear layer andthe outer ganglion cell layer (each analysis was performed separately)and were extracted from the image and transformed into an 8-bitgrayscale image, referred to as a mask. Area and length (to normalizethe data) of the calretinin staining were measured and used to calculatethe area per length of the calretinin staining for each image.

Measurement of Area of the recoverin immunostaining per length: Onesection per eye, immunohistochemically stained for recoverin, was imagedand analyzed. The area where the images were collected was defined asregion 1, an area near the injection site. Up to eight images werecollected from each section. Using Image-Pro Plus, three AOIs weredefined as: all recoverin-positive areas, the inner nuclear layer andthe outer nuclear layer (each analysis was performed separately). EachAOI was extracted from the image and transformed into an 8-bit grayscaleimage, referred to as a mask. Area and length (to normalize the data) ofthe recoverin staining within each AOI was measured and used tocalculate the area per length of the recoverin staining for each image.

Results

Identification of umbilicus-derived cells in day 1 post injection:Umbilicus-derived cell injected (right) eyes from day 1 post-injectionwere sectioned for the purposes of identifying injectedumbilicus-derived cells to confirm their placement in the eye. Usingthis method, injected human cells were positively identified usingimmunohistochemistry for human nuclear matrix antigen (NuMA, FIG. 12).

Umbilicus-derived cell retention in the RCS rat eyes: Umbilicus-derivedcell retention in the RCS eyes was investigated using RT-PCR for ahuman-specific antigen, β2 microglobulin (β2M) mRNA. Total RNA frominjected eyes was amplified using β2M specific primers. Values areconverted to cell number using a standard curve generated using totalRNA isolated from eyes injected intravitreally with known numbers ofumbilicus-derived cells. Despite variability between specimens, eyes atall time points had detectable levels of β2M mRNA. This indicated thatthe umbilicus-derived cells were retained in the RCS eye through thecourse of the study, although the cell number decline substantially byDay 60, with only approximately 10% of the injected cells detected (FIG.13).

Morphometry of the outer nuclear layer: H&E-stained sections of bothcontrol (uninjected) and umbilicus-derived cell-injected eyes from thefollowing time points were examined: 7, 14, 30 and 60 dayspost-injection (FIG. 14). The earliest time point in which ONLdegeneration could be detected in control eyes was Day 30. At this timepoint, the effect of umbilicus-derived cell injection was subtle.However, at Day 60, the effect of umbilicus-derived cell injection wasevident. In control eyes, the ONL had thinned to a discontinuous layer1-2 nuclei in thickness. In the eyes that had received a subretinalinjection of umbilicus-derived cells, the ONL was approximately 3-4nuclei in thickness near the injection site region, looking comparableto the ONL in the Day 30 specimens. This suggested a preservation of theONL by umbilicus-derived cells.

Eyes from day 60 post-injection were evaluated for the effect ofumbilicus-derived cells on outer nuclear layer (ONL) thickness, as ameasure of photoreceptor rescue. Images were taken at 60× magnificationfrom two regions of the retina, near and far from the injection site inH&E stained sections (see FIG. 11). Two eyes were excluded from analysisdue to poor morphology and loss of the majority of the retina,presumably caused by inadequate fixation. The ONL was visibly thicker inumbilicus-derived cell injected eyes compared with control (uninjected)eyes in both regions examined (FIG. 15A-D). Analysis of the images usingmorphometry supported this observation: the area per length occupied byONL nuclei was 7.5 times higher near the injection site, 2.6 timeshigher in areas far from the injection site in umbilicus-derived cellinjected eyes compared with control eyes, and 5 times higher overallwhen the results from both regions were combined, suggesting that theumbilicus-derived cells are preserving photoreceptors in the RCS rat(FIGS. 15E and F). This also shows that the effect is greatest local tothe injection site, however there is an overall effect ofumbilicus-derived cell injection.

Image analysis of rhodopsin immunostaining: Eyes from day 60post-injection were evaluated for the effect of umbilicus-derived cellson expression of rhodopsin using immunohistochemistry and imageanalysis. As with the ONL analysis, a visible difference could be seenin the level of rhodopsin immunostaining in umbilicus-derived cellinjected eyes compared with control eyes (FIGS. 16A and B). Images at60× magnification were acquired from regions near the injection site ofthese eyes. For quantitation of rhodopsin immunostaining, the saturationchannel of the color model HSI was extracted from the RGB image.Saturation refers to the dominance of hue in the color. A dominant hueis considered a pure color and a less dominant hue is a lighter shade ofa pure color, for instance pink is red with a low degree of saturationor dominance. The saturation value, or amount of rhodopsin staining,translates to how densely packed the rods and cones are within theneuroepithelial layer. A highly saturated area is most likely an area oftightly packed rods and cones. Near the injection site region, therhodopsin immunostaining was 10 fold higher in umbilicus-derived cellinjected eyes compared with controls (FIG. 16C), translating to moredensely packed rod outer segments containing rhodopsin and indicatingpreservation of rod photoreceptors.

Image analysis of calreticulin immunostaining: Eyes from day 60post-injection were evaluated for the effect of umbilicus-derived cellson expression of calretinin using immunohistochemistry and imageanalysis. Unlike the ONL and rhodopsin immunostaining, there was not amarked difference visible in the images of calretinin immunostaining incontrol and umbilicus-derived cell injected eyes, however uponquantitation there was a small but statistically significant differencebetween the two groups. Images at 60× magnification were acquired fromregions near the injection site of these eyes (FIGS. 17A and B).Quantitation of calretinin immunostaining was performed in the followinglayers: inner nuclear layer (INL), inner plexiform layer (IPL) andretinal ganglion cell layer (GCL) together, and the INL and GCLindividually. Near the injection site region, overall calretininimmunostaining in all 3 layers was 1.2 fold higher in umbilicus-derivedcell injected eyes compared with controls. In the GCL, there was nodifference between control eyes and umbilicus-derived cell-injectedeyes, however there was a 1.5 fold increase in calretinin immunostainingin the INL of umbilicus-derived cell-injected eyes compared to controls(FIGS. 17C and D), indicating preservation of calretinin-expressingcells in the INL.

Image analysis of recoverin immunostaining: Eyes from day 60post-injection were evaluated for the effect of umbilicus-derived cellson expression of recoverin using immunohistochemistry and imageanalysis. Images at 60× magnification were acquired from regions nearthe injection site of these eyes (FIGS. 18A and B). Quantitation ofrecoverin immunostaining was performed on the inner nuclear layer (INL)and the outer nuclear layer (ONL). Near the injection site region, therecoverin immunostaining was 2 fold higher in the INL and 7.9 foldhigher in the ONL of the umbilicus-derived cell injected eyes comparedwith controls (FIGS. 18C and D), indicating preservation ofrecoverin-expressing cells.

Discussion

Umbilicus-derived cell injection into the subretinal space of RCS ratshas been demonstrated to sustain visual function for several monthsafter injection into the subretinal space of RCS rats (Lund et al.2007). It has also been shown to preserve photoreceptor nuclei in theONL, however this was not done quantitatively. The goal of this studywas to quantitate the effects of umbilicus-derived cell injection on theretina using morphometry. To analyze both ONL thickness as well asquantitation of various retinal cell types using immunohistochemistry,it was crucial to orient the eyes identically in the paraffin block.Additionally, it was necessary to obtain sections near themidline/central plane of the eye, so that the retinal layers to beanalyzed would not appear artificially thick due to taking a slicethrough the curvature of the eye. Great care was taken to attain both ofthese requirements. By using such a careful approach in maintainingorientation of the eyes during processing, embedding and sectioning, thelocation of the injection site region was known in every eye.

Only the 60 days post-injection time point in this study was selectedfor morphometric analysis, as this time point clearly showed an effectof the injected hUTC on ONL thickness in H&E stained sections. Theeffect of hUTC injection on the ONL was evaluated morphometrically. TheONL contains the cell bodies of the photoreceptor cells, and isordinarily packed with nuclei arranged in 10-12 rows. In retinaldegenerative diseases such as retinitis pigmentosa, photoreceptor celldeath results in a thinning of the ONL to approximately 1 layer ofnuclei in thickness. There was a visible difference in ONL thicknessbetween control (uninjected) and umbilicus-derived cell injected eyes 60days post-injection, and this difference was supported morphometrically.Areas both near and far from the injection site were significantlyincreased in umbilicus-derived cell injected eyes, suggestingumbilicus-derived cell injected animals preserved greater numbers ofnucleated cells in the ONL compared with control animals, thusphotoreceptor rescue in these animals. This effect was greatest in theregion near the injection site; therefore this region was selected foranalysis of rhodopsin, calretinin and recoverin immunostaining.

Rhodopsin immunostaining in the neuroepithelial layer was also visiblyincreased in the eyes that received injection of umbilicus-derivedcells. The neuroepithelial layer contains the rods and cones' outersegments, which are specialized processes of the photoreceptor neuronslocated in the ONL. Rod outer segments (ROS) convert and amplify thelight signal. Mammalian ROS are packed with stack of 1000-2000 flatteneddisks that are formed by invaginations of the plasma membrane. The discsof the ROS are responsible for trapping photons and express a high levelof rhodopsin, also known as visual purple. Rhodopsin is also expressedat a lower level on the plasma membrane. ROS are renewed and shed by therod photoreceptors daily, and the shed ROS are phagocytosed and recycledby the RPE. Highly saturated rhodopsin immunostaining was significantlyincreased in umbilicus-derived cell injected animals compared withcontrols, indicating the presence of greater numbers of tightly packedrhodopsin-expressing ROS in the neuroepithelial layer of the retina.This further suggests the rescue/preservation of rod photoreceptorscontaining functional outer segments and is consistent with the resultthat there is photoreceptor rescue in the ONL.

Immunostaining for the calcium-binding protein, calretinin, was alsoevaluated. Calretinin is expressed in neurons of the central nervoussystem, but its precise function has not yet been elucidated. In therat, calretinin expression has been demonstrated in amacrine cellslocated in the INL, retinal ganglion cells in the GCL, and labels 3bands in the IPL, the site for synaptic junctions between the INL andGCL. All three layers staining positively for calretinin werequantitated. While there was not an impressive difference that could bedetected visually in the calretinin staining between control andumbilicus-derived cell injected eyes, there was a small butstatistically significant increase found morphometrically. This 1.2 foldincrease in overall calretinin immunostaining suggested a preservationof calretinin-expressing cells in the retina. Quantitation of calretininimmunostaining was subsequently performed on the INL and GCLindividually to determine if this effect was specific to a particularlayer of the retina. There was no difference in the calretinin stainingin the GCL, however there was a 1.5 fold increase in staining in theINL, suggesting a preservation of calretinin-expressing cells in theinner nuclear layer, possibly amacrine cells.

Recoverin immunostaining was performed on the day 60 specimens.Recoverin is a calcium-binding protein localized to photoreceptor cellbodies in the outer nuclear layer, as well as midget cone bipolar cellsin the inner nuclear layer. Recoverin immunostaining was visiblyincreased in umbilicus-derived cell-injected eyes compared withcontrols, suggesting a preservation of one or more cell types.Morphometry was performed on recoverin staining in the INL and ONL.There was a statistically significant increase in recoverinimmunostaining in umbilicus-derived cell injected eyes compared withcontrols in both layers evaluated, suggesting a preservation ofphotoreceptors in the ONL and of recoverin-expressing cells in the INL,possibly cone bipolar cells.

Taken together, the increase in ONL thickness, rhodopsin, calretinin,and recoverin immunostaining in umbilicus-derived cell-injected eyescompared with controls suggest preservation in the structure of theretina through rescue of photoreceptors, functional rod outer segmentsand possibly bipolar neurons responsible for relaying information fromthe retina to the brain. These results provide support for thefunctional findings that umbilicus-derived cell subretinal injectionpreserves vision in dystrophic RCS rats. The mechanism of action ofumbilicus-derived cells remains to be elucidated. There has been noevidence of differentiation of umbilicus-derived cells intophotoreceptors or other retinal cell types. In fact, only a smallfraction (less than 10%) of injected umbilicus-derived cells aredetectable by quantitative RT-PCR for a human-specific gene 60 dayspost-injection. If the cells do not survive over the course of time inwhich there is a therapeutic effect, perhaps the mechanism is that thecells secrete a neurotrophic factor which slows the apoptotic wave inthe photoreceptor population, thereby allowing for preservation of notonly photoreceptors, but their supporting cells as well.Umbilicus-derived cells are currently being characterized in vitro toaddress secretory factors and mechanistic studies are in progress.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

TABLE 1-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Cells Cell Enzyme Digest Isolated ExpansionCollagenase X X Dispase + (>10 h) + Hyaluronidase X XCollagenase:Dispase ++ (<3 h) ++ Collagenase:Hyaluronidase ++ (<3 h) +Dispase:Hyaluronidase + (>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++ Key: + = good, ++ = very good, +++ = excellent, X = nosuccess under conditions tested

TABLE 1-2 Isolation and culture expansion of postpartum cells undervarying conditions: Growth Condition Medium 15% FBS BME Gelatin 20% O2Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7DMEM-Lg N (2%) Y N (Fibrone) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N (Fibrone)N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N(Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N (5%)EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N (Fibrone) Y PDGF/VEGF 16DMEM-Lg N (2%) N N (Fibrone) N (5%) PDGF/VEGF

TABLE 2-1 Growth characteristics for different cell populations grown tosenescence Total Population Total Cell Cell Type Senescence DoublingsYield MSC 24 d 8 4.72 E7 Adipose 57 d 24  4.5 E12 Fibroblasts 53 d 262.82 E13 Umbilicus 65 d 42 6.15 E17 Placenta 80 d 46 2.49 E19

TABLE 2-2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 till senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 d 43.68 2.59 E11 Umbilicus (P10) 80 d 53.6 1.25 E14 Placenta(P10) 60 d 32.96 6.09 E12

TABLE 3-1 Culture Media Added fetal bovine Culture Medium Supplier serum% (v/v) DMEM low glucose Gibco Carlsbad CA 0, 2 10 DMEM high glucoseGibco 0, 2 10 RPMI 1640 Mediatech, Inc. 0, 2 10 Herndon, VA Cellgro-free (Serum-free, Mediatech, Inc. — Protein-free Ham's F10Mediatech, Inc. 0, 2 10 MSCGM (complete with Cambrex, 0, 2 10 serum)Walkersville, MD Complete-serum free Mediatech, Inc. — w/albumin GrowthMedium NA — Ham's F12 Mediatech, Inc. 0, 2 10 Iscove's Mediatech, Inc.0, 2 10 Basal Medium Eagle's Mediatech, Inc. DMEM/F12 (1:1) Mediatech,Inc. 0, 2 10

TABLE 6-1 Results of PPDC karyotype analysis Metaphase Metaphase cellscells Number of ISCN Tissue passage counted analyzed karyotypesKaryotype Placenta 22 20 5 2 46, XX Umbilical 23 20 5 2 46, XX Umbilical6 20 5 2 46, XY Placenta 2 20 5 2 46, XX Umbilical 3 20 5 2 46, XXPlacenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0 21 54 46, XY[18]/ 46, XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9 25 5 446, XY[5]/ 46, XX[20] Placenta-N 1 20 5 2 46, XY C1 Placenta-N 1 20 6 446, XY[2]/ C3 46, XX[18] Placenta-N 1 20 5 2 46, XY C4 Placenta-N 1 20 52 46, XY C15 Placenta-N 1 20 5 2 46, XY C20 Key: N—Neonatal aspect;V—villous region; M—maternal aspect; C—clone

TABLE 7.1 Catalog Antibody Manufacture Number CD10 BD Pharmingen (SanDiego, CA) 555375 CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446CD34 BD Pharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen555489 CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BDPharmingen 340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen556002 HLA-A, B, C BD Pharmingen 555553 HLA-A-DR, DP, DQ BD Pharmingen555558 IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE Sigma P-4685

TABLE 9-2 The Euclidean Distances for the Cell Pairs. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

TABLE 9-3 Genes shown to have specifically increased expression in theplacenta-derived cells as compared to other cell lines assayed GenesIncreased in Placenta-Derived Cells NCBI Probe Accession Set ID GeneName Number 209732_at C-type (calcium dependent, AF070642carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 AB015228 family, member A2 206367_at reninNM_000537 210004_at oxidized low density AF035776 lipoprotein(lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE: AF0706424179671, mRNA, partial cds 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein AL136883 DKFZp564F013 204135_atdownregulated in ovarian NM_014890 cancer 1 213542_at Homo sapiens mRNA;cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 9-4 Genes shown to have specifically increased expression in theumbilicus-derived cells as compared to other cell lines assayed GenesIncreased in Umbilicus-Derived Cells NCBI Probe Set Accession ID GeneName Number 202859_x_at interleukin 8 NM_000584 211506_s_at interleukin8 AF043337 210222_s_at reticulon 1 BC000314 204470_at chemokine (C—X—Cmotif) ligand 1 NM_001511 (melanoma growth stimulating activity206336_at chemokine (C—X—C motif) ligand 6 NM_002993 (granulocytechemotactic protein 2) 207850_at chemokine (C—X—C motif) ligand 3NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumor necrosisfactor, alpha-induced NM_006290 protein 3

TABLE 9-5 Genes shown to have decreased expression in umbilicus- andplacenta-derived cells as compared to other cell lines assayed GenesDecreased in Umbilicus- and Placenta-Derived Cells NCBI Probe SetAccession ID Gene name Number 210135_s_at short stature homeobox 2AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_atchemokine (C—X—C motif) ligand 12 U19495.1 (stromal cell-derivedfactor 1) 203666_at chemokine (C—X—C motif) ligand 12 NM_000609.1(stromal cell-derived factor 1) 212670_at elastin (supravalvular aorticAA479278 stenosis, Williams-Beuren syndrome) 213381_at Homo sapiensmRNA; cDNA N91149 DKFZp586M2022 (from clone DKFZp586M2022) 206201_s_atmesenchyme homeobox 2 (growth NM_005924.1 arrest-specific homeo box)205817_at sine oculis homeobox homolog 1 NM_005982.1 (Drosophila)209283_at crystallin, alpha B AF007162.1 212793_at dishevelledassociated activator BF513244 of morphogenesis 2 213488_at DKFZP586B2420protein AL050143.1 209763_at similar to neuralin 1 AL040176 205200_attetranectin (plasminogen binding NM_003278.1 protein) 205743_at archomology three (SH3) and NM_003149.1 cysteine rich domain 200921_s_atB-cell translocation gene 1, NM_001731.1 anti-proliferative 206932_atcholesterol 25-hydroxylase NM_003956.1 204198_s_at runt-relatedtranscription AA541630 factor 3 219747_at hypothetical protein FLJ23191NM_024574.1 204773_at interleukin 11 receptor, alpha NM_004512.1202465_at procollagen C-endopeptidase NM_002593.2 enhancer 203706_s_atfrizzled homolog 7 (Drosophila) NM_003507.1 212736_at hypothetical geneBC008967 BE299456 214587_at collagen, type VIII, alpha 1 BE877796201645_at tenascin C (hexabrachion) NM_002160.1 210239_at iroquoishomeobox protein 5 U90304.1 203903_s_at Hephnestin NM_014799.1 205816_atintegrin, beta 8 NM_002214.1 203069_at synaptic vesicle glycoprotein 2NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis, AU147799 cloneMAMMA1001744 206315_at cytokine receptor-like factor 1 NM_004750.1204401_at potassium intermediate/small NM_002250.1 conductancecalcium-activated channel, subfamily N, member 4 216331_at integrin,alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1 213125_atDKFZP586L151 protein AW007573 202133_at transcriptional co-activatorAA081084 with PDZ-binding motif (TAZ) 206511_s_at sinc oculis homeoboxhomolog 2 NM_016932.1 (Drosophila) 213435_at KIAA1034 protein AB028957.1206115_at early growth response 3 NM_004430.1 213707_s_at distal-lesshomeo box 5 NM_005221.3 218181_s_at hypothetical protein FLJ20373NM_017792.1 209160_at aldo-keto reductase family 1, AB018580.1 member C3(3-alpha hydroxysteroid dehydrogenase, type II) 213905_x_at BiglycanAA845258 201261_x_at Biglycan BC002416.1 202132_at transcriptionalco-activator AA081084 with PDZ-binding motif (TAZ) 214701_s_atfibronectin 1 AJ276395.1 213791_at Proenkephalin NM_006211.1 205422_s_atintegrin, beta-like 1 (with NM_004791.1 EGF-like repeat domains)214927_at Homo sapiens mRNA full length AL359052.1 insert cDNA cloneEUROIMAGE 1968422 206070_s_at EphA3 AF213459.1 212805_at KIAA0367protein AB002365.1 219789_at natriuretic peptide receptor AI628360C/guanylate cyclase C (atrionatriuretic peptide receptor C) 219054_athypothetical protein FLJ14054 NM_024563.1 213429_at Homo sapiens mRNA;cDNA AW025579 DKFZp564B222 (from clone DKFZp564B222) 204929_s_atvesicle-associated membrane NM_006634.1 protein 5 (myobrevin)201843_s_at EGF-containing fibulin-like NM_004105.2 extracellular matrixprotein 1 221478_at BCL2/adenovirus E1B 19 kDa AL132665.1 interactingprotein 3-like 201792_at AE binding protein 1 NM_001129.2 204570_atcytochrome c oxidase subunit NM_001864.1 VIIa polypeptide 1 (muscle)201621_at neuroblastoma, suppression of NM_005380.1 tumorigenicity 1202718_at insulin-like growth factor NM_000597.1 binding protein 2, 36kDa

TABLE 9-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. Genes increasedin fibroblasts dual specificity phosphatase 2 KIAA0527 protein Homosapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic,intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G)inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 9-7 Genes that were shown to have increased expression in theICBM-derived cells as compared to the other cell lines assayed, GenesIncreased In ICBM Cells cardiac ankyrin repeat protein MHC class Iregion ORF integrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-induced protein44 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) keratin associated protein 1-1 hippocalcin-like 1 jagged 1(Alagille syndrome) proteoglycan 1, secretory granule

TABLE 9-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. Genes Increased InMSC Cells interleukin 26 maltase-glucoamylase (alpha-glucosidase)nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murineosteosarcoma viral oncogene homolog hypothetical protein DC42 nuclearreceptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viraloncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cellline derived transforming sequence potassium channel, subfamily K,member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNAFLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, cloneLIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc fingerprotein 51) zinc finger protein 36, C3H type, homolog (mouse)

TABLE 10-1 Primers used Primer name Primers Oxidized LDL S: 5′-GAGAAATCCAAAGAGCAAATGG-3′ receptor (SEQ ID NO: 1)A: 5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO: 2) ReninS: 5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO: 3)A: 5′-GAATTCTCGGAATCTCTGTTG-3′ (SEQ ID NO: 4) ReticulonS: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO: 5)A: 5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ′ ID NO: 6) Interleukin-8S: 5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO: 7)A: 5′-CTTCAAAAACTTCTCCACAACC-3′ (SEQ ID NO: 8) Chemokine 5: 5′-CCCACGCCACGCTCTCC-3′ (CXC) ligand 3 (SEQ ID NO: 9)A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO: 10)

TABLE 10-2 IL-8 protein amount measured by ELISA Cell type IL-8 hFibroND Placenta Isolate 1 ND Umb Isolate 1 2058.42 ± 144.67 Placenta Isolate2 ND Umb Isolate 2 2368.86 ± 22.73  Placenta Isolate3 (normal O₂) 17.27± 8.63 Placenta Isolate 3 (low O₂, W/O BME) 264.92 ± 9.88  Results ofthe ELISA assay for interleukin-8 (IL-8) performed on placenta-andumbilicus-derived cells as well as human skin fibroblasts. Values arepresented here are picograms/million cells, n = 2, sem. ND: Not Detected

TABLE 11-1 Antibodies Catalog Antibody Manufacturer Number HLA-DRDPDQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA)557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen(San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappaSigma (St. Louis, MO) P-4685

TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPMfor Proliferation Assay Analytical Replicates number Culture System 1 23 Mean SD CV Plate ID: Plate 1 IM03-7769 Proliferation baseline ofreceiver 79 119 138 112.0 30.12 26.9 Control of autostimulation(Mitomycin C treated autologous cells) 241 272 175 229.3 49.54 21.6 MLRallogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 2092122414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B)664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Controlof autostimulation (Mitomycin C treated autologous cells) 1091 602 524739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C treated)45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin Ctreated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI(cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128124.0 35.17 28.4 Control of autostimulation (Mitomycin C treatedautologous cells) 293 138 508 313.0 185.81 59.4 MLR allogenic donorIM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8MLR with cell line (Mitomycin C treated cell type B) 601 643 4 622.029.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferationbaseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation(Mitomycin C treated autologous cells) 133 120 213 155.3 50.36 32.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 2216818822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell typeB) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferationbaseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic Control ofautostimulation (Mitomycin treated autologous cells) 361 617 304 427.3166.71 39.0 donor) Proliferation baseline of receiver 126 124 143 131.010.44 8.0 Cell line Control of autostimulation (Mitomycin treatedautologous cells) 822 1075 487 794.7 294.95 37.1 type B Plate ID: Plate2 IM03-7773 Proliferation baseline of receiver 908 181 330 473.0 384.0281.2 Control of autostimulation (Mitomycin C treated autologous cells)269 405 572 415.3 151.76 36.5 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 29151 28691 28315 28719.0 418.70 1.5 MLR with cell line(Mitomycin C treated cell type B) 567 732 905 734.7 169.02 23.0 SI(donor) 61 SI (cell line) 2 IM03-7774 Proliferation baseline of receiver893 1376 185 818.0 599.03 73.2 Control of autostimulation (Mitomycin Ctreated autologous cells) 261 381 568 403.3 154.71 38.4 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 53101 42839 48283 48074.3 5134.1810.7 MLR with cell line (Mitomycin C treated cell type B) 515 789 194532.7 247.97 46.6 SI (donor) 59 SI (cell line) 1 IM03-7775 Proliferationbaseline of receiver 1272 300 544 705.3 505.69 71.7 Control ofautostimulation (Mitomycin C treated autologous cells) 232 199 484 305.0155.89 51.1 MLR allogenic donor IM03-7768 (Mitomycin C treated) 2355410523 28965 21014.0 9479.74 45.1 MLR with cell line (Mitomycin C treatedcell type B) 768 924 563 751.7 181.05 24.1 SI (donor) 30 SI (cell line)1 IM03-7776 Proliferation baseline of receiver 1530 137 1046 904.3707.22 78.2 Control of autostimulation (Mitomycin C treated autologouscells) 420 218 394 344.0 109.89 31.9 MLR allogenic donor IM03-7768(Mitomycin C treated) 28893 32493 34746 32044.0 2952.22 9.2 MLR withcell line (Mitomycin C treated cell type B) a a a a a a SI (donor) 35 SI(cell line) 2

TABLE 11-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

TABLE 11-4 Mixed Lymphocyte Reaction Data- Cell Line A (Umbilicus) DPMfor Proliferation Assay Analytical Replicates number Culture System 1 23 Mean SD CV Plate ID: Plate 1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic Control of autostimulation (Mitomycin treated autologouscells) 567 604 374 515.0 123.50 24.0 donor) Proliferation baseline ofreceiver 5101 3735 2973 3936.3 1078.19 27.4 Cell line Control ofautostimulation (Mitomycin treated autologous cells) 1924 4570 21532882.3 1466.04 50.9 type A

TABLE 11-5 Average stimulation index of umbilicus-derived cells and anallogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Recipient UmbilicusPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

TABLE 12-1 ELISA assay results MCP-1 IL-6 VEGF SDF-1α GCP-2 IL-8 TGF-β2Fibroblast 17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta 60 ± 3 41 ±2 ND ND ND ND ND (042303) Umbilicus 1150 ± 74  4234 ± 289 ND ND 160 ± 112058 ± 145 ND (022803) Placenta 125 ± 16 10 ± 1 ND ND ND ND ND (071003)Umbilicus 2794 ± 84  1356 ± 43  ND ND 2184 ± 98  2369 ± 23  ND (071003)Placenta  21 ± 10 67 ± 3 ND ND 44 ± 9 17 ± 9 ND (101503) BME Placenta 77 ± 16 339 ± 21 ND 1149 ± 137 54 ± 2 265 ± 10 ND (101503) 5% O₂, W/OBME (values presented are picograms/milliliter/million cells (n = 2,sem) Key: ND: Not Detected.

TABLE 12-2 SearchLight Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF hFB 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.813 ND U1 57718.4 ND ND1240.0 5.8 559.3 148.7 ND 9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 NDND 1.9  33.6 U3 21850.0 ND ND 1134.5 9.0 195.6  30.8 ND 5.4 388.6 Key:hFB (human fibroblasts), P1 (placenta-derived cells (042303)), U1(umbilicus-derived cells (022803)), P3 (placenta-derived cells(071003)), U3 (umbilicus-derived cells (071003)). ND: Not Detected.

TABLE 12-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4  4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.511.6 21.4 ND  4.8 10515.9 Key: hFB (human fibroblasts), P1(placenta-derived PPDC (042303)), U1 (umbilicus-derived PPDC (022803)),P3 (placenta-derived PPDC (071003)), U3 (umbilicus-derived PPDC(071003)). ND: Not Detected.

TABLE 13-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA Human Nestin 1:100Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA Tyrosine 1:1000 Chemicon hydroxylase(TH) GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemicon alpha -alpha-smooth 1:400 Sigma muscle actin Human nuclear 1:150 Chemiconprotein (hNuc)

TABLE 13-2 Summary of Conditions for Two-Stage Differentiation ProtocolA B COND. # PRE-DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F (20 ng/ml) +E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F (20ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 μM)3 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 μM) 4 NPE + F (20ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F (20ng/ml) + E (20 ng/ml) Growth Medium 6 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + MP52 (20 ng/ml) 7 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + MP52 (20 ng/ml) 10 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + MP52 (20 ng/ml) 13 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + E (20 ng/ml)NPE + MP52 (20 ng/ml) 16 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7(20 ng/ml) 17 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)

TABLE 14-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA

TABLE 15-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase 1:1000 Chemicon (TH)GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

TABLE 15-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond.5] TuJ1  8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

TABLE 18-1 ERG data a-wave mixed b-wave cone b-wave % rod contributionGroup Untreated Treated Untreated Treated Untreated Treated UntreatedTreated Sham 60 d 0 0 7 ± 9 0 23 ± 5  12 ± 16 N/A N/A 1 (n = 4) 60 d 020 ± 20 1.5 ± 2   81 ± 72 7 ± 7 50 ± 19 N/A 30 3 (n = 6) 60 d 0 27 ± 1118 ± 13 117 ± 67  28 ± 11 55 ± 25 6 ± 7 49 ± 16 3 (n = 6) 90 d 0 15 ± 7 0 37 ± 15 7 ± 5 16 ± 11 0 58 ± 39 N.B. Sham = control (medium only), 1 =Placental cell transplant, 3 = Umbilical cell transplant

TABLE 19.1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase 1:1000 Chemicon (TH)GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

TABLE 19-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond.5] TuJ1  8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

TABLE 22-1 TUNEL Area Analysis at Post Natal Day 29 P29 (8 days posttreatment) Group DAPI+ area (pixels) % TUNEL+ Congenic untreated(healthy) 373,812 ± 12,832  0.2 ± 0.2% Dystrophic-untreated — —Dystrophic-sham (vehicle only) 222,016 ± 23,242 16.0 ± 2.3%Dystrophic-hUTC injected 229,666 ± 6,383  6.6 ± 0.5% (20,000 cells)

TABLE 25-1 Integrin hUTC ARPE-19 Fetal RPE * α1 − − + α2 + − + α3 + + +α4 + + + α5 + + + β1 + + + α5 β1 − − + α2 β1 + + +

TABLE 26-1 Reagents used in first step in SuperScript III First-StrandSynthesis: Component: 50 μM 10 mM DEPC-treated Total RNA oligo(dT)20dNTP mix water Amount: 2 μg 2 μL 2 μL To raise volume to 20 μL

TABLE 26-2 Reagents used in cDNA Synthesis Mix: Component: 10X RT 25 mM0.1 M RNaseOUT. SuperScript. buffer MgCl₂ DTT (40 U/μl) III RT Amount: 4μL 8 μL 4 μL 2 μL 2 μL

TABLE 26-3 Reagents used in Master Mix for the RT-PCR reaction: TaqMan ®Component: Gene Expression Master Mix cDNA dH₂0 Assays Amount: 10 μL 3μL 6 μL 1 μL

TABLE 26-4 TaqMan ® gene expression assays for real time RT-PCRanalysis: Taqman Gene Abbreviation Name Expression Assay Proposedfunction in the eye CRALBP Cellular retinaldehyde-binding proteinHS00165632_m1 Regeneration of visual pigment in RPE MERTK c-mer protooncogene tyrosine kinase HS00179024_m1 ROS phagocytosis (ROSinternalization) GAS6 growth arrest specific-6 (ligand for MERTK)Hs00181323_m1 ROS phagocytosis (ROS internalization) CD36 lipidscavenger receptor CD36 HS00169627_m1 ROS phagocytosis (ROSinternalization) INTaV Integrin alpha V Hs00233808_m1 ROS phagocytosis(ROS binding) INTB5 Integrin beta 5 Hs00174435_m1 ROS phagocytosis (ROSbinding) CATD Cathepsin D Hs00157205_m1 ROS degradation (lysosomalenzyme) CFH Complement Factor H HS00164830_m1 Mediates inflammation

TABLE 26-5 Calculated □CT values for each target. Values were normalizedto GAPDH (endogenous control). Fold change is 2^(□Ct). Smaller □CTvalues represent increased gene expression. MERTK CRALBP GAS6 INTAVINTB5 CD36 CATHD CFH Untreated 16.6 ± 1.2   21 ± 3.9 3.5 ± 0.3 5.5 ± 2.22.9 ± 0.4 14.4 ± 1.0 2.1 ± 1.5 8.2 ± 0.2 hUTC 11.8 ± 0.6 14.3 ± 2.6 5.0± 1.1 2.5 ± 0.5 3.0 ± 1.5 15.3 ± 1.3 0.3 ± 0.6 2.8 ± 0.2 Treament

TABLE 27-4 Trophic Factor Secretion by hUTC Sample hUTC hUTC hFlb. hFib.FGF-b pg/ml/million 64.06 74.00 7.43 7.64 HGF pg/ml/million 980.23703.24 37.76 30.54 KGF pg/ml/million 50.78 41.15 81.28 63.43 VEGFpg/ml/million 118.00 90.80 202.52 165.96 GRO-a pg/ml/million 3758.522398.48 795.43 760.72 MCP1 pg/ml/million 948.56 625.71 218.83 182.95GMCSF pg/ml/million 400.16 267.54 129.11 150.81 IL6 pg/ml/million 103.5769.23 75.39 55.46 IL8 pg/ml/million 7308.60 3832.09 3843.18 2781.39 TNFapg/ml/million 21.72 6.91 6.91 6.91 B-NGF pg/ml/million 5.16 1.18 10.068.28 BDNF pg/ml/million 525.38 432.08 220.41 191.69 CNTF pg/ml/million102.11 74.58 55.06 10.00 NT-3 pg/ml/million 33.29 16.78 22.87 21.38NT-proBN pg/ml/million 24.14 21.91 14.92 12.56 TGFb pg/ml/million 662.45718.24 1144.50 298.62

TABLE 27-5 IGF Secretion buy hUTC IGF(ng/ml/million) SD hUTC 43529.4143.53 Fib 24759.26 24.70

What is claimed is:
 1. A method for treating damage to the optic nervein a patient having glaucoma, the method comprising administering to thedamaged optic nerve isolated umbilicus-cells isolated from humanumbilical cord tissue substantially free of blood, in an amounteffective to treat the damage, wherein the cells are capable ofself-renewal and expansion in culture, have the potential todifferentiate into cells of at least a neural phenotype, maintain anormal karyotype upon passaging, and have the following characteristics:a) potential for at least 40 population doublings in culture; b)production of CD10, CD13, CD44, CD73, and CD90; c) lack of production ofCD31, CD34, CD45, CD117 and CD141, and d) increased expression of genesencoding interleukin 8 and reticulon 1 relative to a human cell that isa fibroblast, a mesenchymal stem cell, or an iliac crest bone marrowcell.
 2. The method of claim 1, wherein the umbilicus cells are positivefor HLA-A,B,C, and negative for HLA-DR,DP,DQ.
 3. The method of claim 1,wherein the umbilicus cells are expanded in culture prior toadministering to the patient's eye.
 4. The method of claim 1, whereinthe umbilicus cells are administered with at least one other agent. 5.The method of claim 4, wherein the at least one other agent isadministered simultaneously with, or before, or after, the umbilicuscells.
 6. The method of claim 1, wherein the umbilicus cells areadministered through a cannula or from a device inserted in thepatient's eye.
 7. The method of claim 1, wherein the umbilicus cells areadministered by insertion of a matrix or scaffold containing the cells.8. A method for reducing the loss of photoreceptor cells in a patientwith a retinal degeneration, the method comprising administering to theinterior of the patient's eye isolated umbilicus cells isolated fromhuman umbilical cord tissue substantially free of blood, in an amounteffective to reduce the loss of photoreceptor cells, wherein theumbilicus cells are capable of self-renewal and expansion in culture,have the potential to differentiate into cells of at least a neuralphenotype, maintain a normal karyotype upon passaging, and have thefollowing characteristics: a) potential for at least 40 populationdoublings in culture; b) production of CD10, CD13, CD44, CD73, and CD90;c) lack of production of CD31, CD34, CD45, CD117, and CD141, and d)increased expression of genes encoding interleukin 8 and reticulon 1relative to a human cell that is a fibroblast, a mesenchymal stem cell,or an iliac crest bone marrow cell.
 9. The method of claim 8, whereinthe umbilicus cells are positive for HLA-A,B,C, and negative forHLA-DR,DP,DQ.
 10. The method of claim 8, wherein the umbilicus cells areexpanded in culture prior to administering to the patient's eye.
 11. Themethod of claim 8, wherein the retinal degeneration is age-relatedmacular degeneration.
 12. The method of claim 8, wherein the loss ofphotoreceptor cells is reduced by inhibiting the apoptosis of thephotoreceptor cells.
 13. The method of claim 8, wherein the loss ofphotoreceptor cells is reduced by stimulating the phagocytosis of shedphotoreceptor fragments.
 14. The method of claim 13, wherein trophicfactors secreted by the umbilicus cells stimulate retinal pigmentepithelial (RPE) cells to phagocytose the shed photoreceptor fragments.15. The method of claim 13, wherein the umbilicus cells stimulate thephagocytosis of shed photoreceptor fragments by retinal pigmentepithelium (RPE) cells.
 16. The method of claim 8, wherein the umbilicuscells are administered with at least one other agent.
 17. The method ofclaim 16, wherein the at least one other agent is administeredsimultaneously with, or before, or after, the umbilicus cells.
 18. Themethod of claim 8, wherein the umbilicus cells are administered througha cannula or from a device inserted in the patient's eye.
 19. The methodof claim 8, wherein the umbilicus cells are administered by insertion ofa matrix or scaffold containing the umbilicus cells.
 20. The method ofclaim 11, wherein the age-related macular degeneration is dryage-related macular degeneration.